Methods of treating chronic disorders with complement inhibitors

ABSTRACT

In some aspects, the invention provides methods of treating a subject in need of treatment for a chronic complement-mediated disorder. In some aspects, the invention provides methods of treating a subject in need of treatment for a Th17-associated disorder. In some aspects, the invention provides methods of treating a subject in need of treatment for a chronic respiratory system disorder. In some aspects, the invention provides methods of administering a complement inhibitor to a subject. In some embodiments, a method of treating a subject comprises administering multiple doses of a complement inhibitor to the subject according to a dosing schedule that leverages the prolonged effect of complement inhibition in chronic respiratory disorders. In some embodiments, a subject has chronic obstructive pulmonary disease. In some embodiments, a subject has asthma.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. national phase application under 35. U.S.C. §371 of international PCT application no. PCT/US2012/043845, filed Jun.22, 2012, whe claims priority to U.S. provisional patent application No.61/499,895, filed Jun. 22, 2011. The entire contents of theseapplications are hereby incorporated by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 17, 2013, isnamed 2008575-0068_SL.txt and is 37,872 bytes in size.

BACKGROUND OF THE INVENTION

Chronic disorders of the respiratory system are significant causes ofmorbidity and mortality whose incidence is increasing worldwide.According to World Health Organization estimates, about 80 millionpeople have moderate to severe chronic obstructive lung disease (COPD),and more than 3 million people died of COPD in 2005 (˜5% of all deathsglobally). COPD was the fifth leading cause of death in 2002, andestimates suggest that it will be the third leading cause of deathworldwide in 2030 unless major risk factors, particularly tobacco use,can be successfully curbed. Asthma is also a significant global healthproblem, affecting an estimated 300 million individuals worldwide. Bothasthma and COPD can have debilitating effects on patients' dailyfunctioning and quality of life, particularly when severe. Thesediseases also represent significant burdens in terms of health carecosts and lost productivity.

Pharmacological therapies such as bronchodilators and corticosteroidsare widely used in the treatment of asthma and COPD. However, asignificant proportion of patients experience persistent symptomsdespite such interventions. Furthermore, these agents can be associatedwith significant side effects. There is a need for additionalpharmacological therapies for treating disorders affecting therespiratory system.

SUMMARY OF THE INVENTION

The invention provides, among other things, methods of treating achronic complement-mediated disorder, the methods comprisingadministering a complement inhibitor to a subject in need of treatmentfor the disorder. In some aspects, the invention provides methods oftreating a chronic disorder of the respiratory system, the methodscomprising administering a complement inhibitor to a subject in need oftreatment for the disorder. In some embodiments, the disorder is asthma.In some embodiments, the disorder is COPD. Certain aspects of theinvention are based at least in part on the recognition that complementinhibitors exhibit a prolonged duration of effect in the treatment ofchronic complement-mediated disorders, e.g., chronic complement-mediateddisorders of the respiratory system, such as asthma or COPD. Forexample, in some embodiments, the duration of action of a complementinhibitor for significantly reducing one or more manifestation(s) of achronic complement-mediated disorder, e.g., a chronic respiratorydisorder, is greater than the duration of action of the complementinhibitor for substantially inhibiting plasma complement activationcapacity when administered intravenously.

In some aspects, the invention provides a method of treating a subjectin need of treatment for a chronic respiratory disorder or other chroniccomplement-mediated disorder, the method comprising administeringmultiple doses of a complement inhibitor to the subject according to adosing schedule in which successive doses are administered on average(i) at least 2 weeks after the plasma concentration of the complementinhibitor decreases to no more than 20% of the maximum plasmaconcentration that was reached after the previous dose; (ii) at least 2weeks after plasma complement activation capacity has returned to atleast 50% of baseline after the previous dose; (iii) at intervals equalto at least 2 times the terminal plasma half-life of the complementinhibitor; or (iv) at intervals at least 3 weeks apart. In someembodiments successive doses of the complement inhibitor areadministered on average (i) between 2 weeks and 6 weeks after the plasmaconcentration of the complement inhibitor decreases to no more than 20%of the maximum plasma concentration that was reached after the previousdose; (ii) between 2 weeks and 6 weeks after plasma complementactivation capacity has returned to at least 50% of baseline after theprevious dose; (iii) at intervals equal to between 2 and 5 times theterminal plasma half-life of the complement inhibitor; or (iv) atintervals between 3 weeks and 6 weeks apart. In some embodimentssuccessive doses of the complement inhibitor are administered on averageat least 4 weeks apart. In some embodiments successive doses of thecomplement inhibitor are administered on average at least 2 weeks afterplasma complement activation capacity has returned to within the normalrange after the previous dose. In some embodiments successive doses ofthe complement inhibitor are administered on average at least 2 weeksafter the plasma concentration of the complement inhibitor decreases tono more than 10% of the maximum plasma concentration that was reachedafter the previous dose. In some embodiments successive doses of thecomplement inhibitor are administered on average at least 2 weeks afterthe plasma concentration of the complement inhibitor decreases to nomore than 5% of the maximum plasma concentration that was reached afterthe previous dose. In some embodiments wherein the dosing schedule isdetermined based at least in part on values of the complement inhibitorplasma concentration, complement inhibitor plasma half-life, and/orplasma complement activation capacity, as measured in a population ofsubjects. In some embodiments the dosing schedule is determined based atleast in part on values of the complement inhibitor plasmaconcentration, complement inhibitor plasma half-life, and/or plasmacomplement activation capacity, of the subject being treated.

In some embodiments of any method comprising dosing, at least 5 dosesare administered.

In some embodiments a subject is in need of treatment for asthma,chronic obstructive pulmonary disease (COPD), or both. In someembodiments a subject is in need of treatment for severe asthma.

In some embodiments a complement inhibitor is administered by therespiratory route. In some embodiments a complement inhibitor isadministered using a nebulizer, metered dose inhaler, or dry powderinhaler. In some embodiments a complement inhibitor is administeredusing a vibrating mesh nebulizer.

In some embodiments a complement inhibitor is administered by theintravenous route.

In some embodiments a complement inhibitor acts on C3 or upstream of C3in the complement cascade. In some embodiments the complement inhibitorinhibits cleavage of C3, C5, or factor B.

In some embodiments a complement inhibitor comprises an antibody,aptamer, peptide, polypeptide, or small molecule.

In some embodiments a complement inhibitor comprises an antibody,aptamer, peptide, polypeptide, or small molecule that binds to C3, C5,factor B, or factor D.

In some embodiments a complement inhibitor comprises a compstatinanalog.

In some embodiments a complement inhibitor comprises a compstatin analogwhose sequence comprises SEQ ID NO: 14, 21, 28, 29, 32, 33, 34, or 36.

In some embodiments a complement inhibitor comprises a compstatin analogwhose sequence comprises any of SEQ ID NOs: 3-41.

In some embodiments a complement-mediated disorder is a Th17-associateddisorder.

In some embodiments any method of treatment comprises detecting a Th17biomarker in the subject or in a sample obtained from the subject. Insome embodiments the Th17 biomarker is detected in a sample comprising abody fluid, wherein the body fluid is optionally selected from blood,BAL fluid, sputum, nasal secretion, or urine or a combination thereof.In some embodiments the biomarker comprises at least one cytokine thatis produced by or promotes formation, survival, or activity of Th17cells. In some embodiments an increased level of the Th17 biomarker ascompared to a reference indicates that the subject is in need of a doseof the complement inhibitor. In some embodiments the reference is withinthe normal range for persons not suffering from the disorder or is abaseline value for the subject when the disorder is well controlled. Insome embodiments the Th17 biomarker is detected prior to administrationof a dose of the complement inhibitor and serves as an indicator thatthe subject is in need of a dose of the complement inhibitor. In someembodiments the biomarker is detected prior to administration of a doseof the complement inhibitor and serves as an indicator that the subjectis in need of a dose of the complement inhibitor, and the methodcomprises administering the complement inhibitor within a predeterminedtime period following detection of the biomarker. In some embodiments apredetermined time period is 1, 2, 3, 4, 5, 6, or 7 days or 2, 3, or 4weeks.

In some aspects, a method of treating a subject in need of treatment fora chronic complement-mediated disorder comprises: (a) administering atleast one dose of a complement inhibitor to the subject; and (b)monitoring the subject for a Th17 biomarker in the subject or in asample obtained from the subject. In some embodiments the method,further comprises: (c) administering at least one additional dose of thecomplement inhibitor to the subject. In some embodiments step (b)comprises detecting a Th17 biomarker in the subject or in a sampleobtained from the subject. In some embodiments step (b) comprisesdetecting an increased level of the biomarker as compared to areference, wherein the increased level indicates that the subject is inneed of a dose of the complement inhibitor. In some embodiments step (b)comprises detecting an increased level of the biomarker as compared to areference, wherein the increased level indicates that the subject is inneed of a dose of the complement inhibitor, and the method furthercomprises (c) administering at least one additional dose of thecomplement inhibitor to the subject. In some embodiments step (b)comprises detecting an increased level of the biomarker as compared to areference, wherein the increased level indicates that the subject is inneed of a dose of the complement inhibitor, and the method furthercomprises (c) administering at least one additional dose of thecomplement inhibitor to the subject within a predetermined time ofdetecting the biomarker. In some embodiments a predetermined time periodis 1, 2, 3, 4, 5, 6, or 7 days or 2, 3, or 4 weeks. In some embodimentsa method further comprises administering an anti-Th17 agent to thesubject.

In some embodiments an anti-Th17 agent comprises an agent that inhibitsformation or activity of Th17 cells. In some embodiments an anti-Th17agent comprises an agent that inhibits the production or activity of acytokine produced by Th17 cells or that promotes formation or activityof Th17 cells. In some embodiments an anti-Th17 agent comprises an agentthat inhibits the production or activity of IL-1β, IL-6, IL-21, IL-22,IL-17, or IL-23. In some embodiments an anti-Th17 agent comprises anantibody, small molecule, aptamer, polypeptide, or RNAi agent. In someembodiments an anti-Th17 agent comprises an antibody, small molecule,aptamer, or polypeptide that binds to IL-1β, IL-6, IL-21, IL-22, IL-17,or IL-23 or binds to receptor for any of the foregoing.

In some aspects, a pharmaceutical composition comprising a complementinhibitor and an anti-Th17 agent is provided. In some embodimentswherein the complement inhibitor inhibits C3 activity or C3 activation.In some embodiments the complement inhibitor comprises a compstatinanalog. In some embodiments wherein the anti-Th17 agent comprises anantibody, small molecule, aptamer, or polypeptide that binds to IL-1β,IL-6, IL-21, IL-22, IL-17, or IL-23 or binds to receptor for any of theforegoing.

In some aspects, a method of treating a complement-mediated disordercomprises administering a composition comprising a complement inhibitorand an anti-Th17 agent to a subject in need thereo.

In some aspects, a method of treating a Th17-associated disordercomprises administering a complement inhibitor and an anti-Th17 agent toa subject in need thereof.

In some aspects, a method of method of disrupting aDC-Th17-B-Ab-C-DCcycle is provided, the method comprising administeringcomprising a complement inhibitor and an anti-Th17 agent to a subject inneed thereof.

In some aspects, a method of treating a Th17-associated disordercomprises administering a complement inhibitor and an anti-Th17 agent toa subject in need thereof.

In some aspects, a method of treating a Th17-associated disordercomprises administering a composition comprising a complement inhibitorand an anti-Th17 agent to a subject in need thereof.

In some aspects, a method of method of disrupting a DC-Th17-B-Ab-C-DCcycle is provided, the method comprising administering comprising acomplement inhibitor and an anti-Th17 agent to a subject in needthereof.

In some embodiments, any of the methods comprises monitoring the subjectfor evidence of a DC-Th17-B-Ab-C-DC cycle.

In some embodiments, any of the methods comprises monitoring the subjectfor evidence of a DC-Th17-B-Ab-C cycle and administering a complementinhibitor, anti-Th17 agent, or composition comprising a complementinhibitor, anti-Th17 agent to the subject based at least in part on aresult of said monitoring.

In some embodiments, any of the methods comprises monitoring the subjectfor a Th17 biomarker.

In some embodiments, any of the methods comprises monitoring the subjectfor a Th17 biomarker and administering a complement inhibitor, anti-Th17agent, or composition comprising a complement inhibitor, anti-Th17 agentto the subject based at least in part on a result of the monitoring.

In some aspects, a method of treating a subject having or at risk of acomplement-mediated disorder, comprises monitoring the subject forevidence of a DC-Th17-B-Ab-C-DC cycle and administering a complementinhibitor to the subject based at least in part on a result of saidmonitoring. In some embodiments the method further comprisesadministering an anti-Th17 agent to the subject.

In some aspects, a method of treating a subject having or at risk of acomplement-mediated disorder, comprises monitoring the subject forevidence of a DC-Th17-B-Ab-C-DC cycle and administering a complementinhibitor and an anti-Th17 agent to the subject based at least in parton a result of said monitoring.

In some aspects, a method of treating a subject having or at risk of aTh17-associated disorder, the method comprising monitoring the subjectfor evidence of a DC-Th17-B-Ab-C-DC cycle and administering a complementinhibitor to the subject based at least in part on a result of saidmonitoring.

In some embodiments the method further comprises administering ananti-Th17 agent to the subject.

In some aspects, a method of treating a subject having or at risk of aTh17-associated disorder is provided, the method comprising monitoringthe subject for evidence of a DC-Th17-B-Ab-C-DC cycle and administeringa complement inhibitor and an anti-Th17 agent to the subject based atleast in part on a result of said monitoring. In some embodiments thecomplement inhibitor inhibits C3 activity or C3 activation. In someembodiments the complement inhibitor comprises a compstatin analog.

In some embodiments of a composition or method relating at least in partto an anti-Th17 agent, the anti-Th17 agent comprises an antibody, smallmolecule, aptamer, or polypeptide that binds to IL-1β, IL-6, IL-21,IL-22, IL-17, or IL-23 or binds to receptor for any of the foregoing.

In some embodiments of any method comprising monitoring a subject forevidence of a DC-Th17-B-Ab-C-DC cycle, such monitoring comprisesassessing a Th17-associated biomarker in the subject or in a sampleobtained from the subject.

In some embodiments of any method comprising monitoring a subject, themonitoring occurs approximately every 1-2 weeks, 2-4 weeks, orapproximately every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.

In some embodiments of any method comprising administering a complementinhibitor, anti-Th17 agent, or both, administration occurs within nomore than 1, 2, 3, 4, 5, 6, or 7 days or 2, 3, or 4 weeks of havingdetected evidence of a DC-Th17-B-Ab-C-DC cycle or increased level of aTh17-associated biomarker.

In some aspects, a method of treating a subject in need of treatment forAMD is provided, the method comprising administering an anti-IL-23 agentto the subject. In some embodiments the agent is administered locally tothe eye, e.g., by intravitreal injection. In some embodiments of thesubject has dry AMD.

All articles, books, patent applications, patents, other publications,websites, and databases mentioned in this application are incorporatedherein by reference. In the event of a conflict between thespecification and any of the incorporated references the specification(including any amendments thereto) shall control. Unless otherwiseindicated, art-accepted meanings of terms and abbreviations are usedherein.

BRIEF DESCRIPTION OF THE DRAWING

FIGS. 1-11 are plots that show concentrations in broncheoalveolar lavage(BAL) fluid of the indicated cytokines, measured in samples obtainedfrom individual cynomolgus monkeys at the indicated time points prior toor following Ascaris suum challenges 0, 1, and 2. Controlanimals(triangles); budesonide-treated animals (+); CA-28-treated animals(circles). Plots of mean cytokine concentration at each time point aresuperimposed and shown as continuous lines to more clearly depictchanges over the 24 hour time period.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

I. Definitions

As used herein, the term “antibody” encompasses antibodies and antibodyfragments comprising an antigen binding site. Antibodies useful incertain embodiments of the invention could originate from or be derivedfrom various species, e.g., human, non-human primate, rodent (e.g.,mouse, rat, rabbit), goat, chicken, and/or could be of various antibodyclasses, e.g., the human classes: IgG (e.g., IgG1, IgG2, IgG3, IgG4),IgM, IgA, IgD, and IgE. An antibody fragment (Fab) can be, for example,a Fab′, F(ab′)₂, scFv (single-chain variable) or other fragment thatretains or contains an antigen binding site. See, e.g., Allen, T.,Nature Reviews Cancer, Vol. 2, 750-765, 2002, and references therein.Antibodies known in the art as diabodies, minibodies, or nanobodies canbe used in various embodiments. Bispecific or multispecific antibodiesmay be used in various embodiments. The heavy and light chain of IgGimmunoglobulins (e.g., rodent or human IgGs) contain four frameworkregions (FR1 through FR4) separated respectively by threecomplementarity determining regions (CDR1 through CDR3). The CDRs,particularly the CDR3 regions, especially the heavy chain CDR3, arelargely responsible for antibody specificity. An antibody may be achimeric antibody in which, for example, a variable domain of rodentorigin or non-human primate origin is fused to a constant domain ofhuman origin, or a “humanized” antibody in which some or all of thecomplementarity-determining region (CDR) amino acids that constitute anantigen binding site (sometimes along with one or more framework aminoacids or regions) are “grafted” from a rodent antibody (e.g., murineantibody) or phage display antibody to a human antibody, thus retainingthe specificity of the rodent or phage display antibody. Thus, humanizedantibodies may be recombinant proteins in which only the antibodycomplementarity-determining regions are of non-human origin. It will beappreciated that the alterations to antibody sequence that are involvedin the humanization process are generally carried out through techniquesat the nucleic acid level, e.g., standard recombinant nucleic acidtechniques. In some embodiments only the specificity determiningresidues (SDRs), the CDR residues that are most crucial in theantibody-ligand interaction, are grafted. The SDRs may be identified,e.g., through use of a database of the three-dimensional structures ofthe antigen-antibody complexes of known structures or by mutationalanalysis of the antibody-combining site. In some embodiments an approachis used that involves retention of more CDR residues, namely grafting ofso-called “abbreviated” CDRs, the stretches of CDR residues that includeall the SDRs. See, e.g., Kashmiri, S V, Methods. 36(1):25-34 (2005), forfurther discussion of SDR grafting. See, e.g., Almagro J C, Fransson J.Humanization of antibodies. Front Biosci. 13:1619-33 (2008) for reviewof various methods of obtaining humanized antibodies. It will beunderstood that “originate from or derived from” refers to the originalsource of the genetic information specifying an antibody sequence or aportion thereof, which may be different from the species in which anantibody is initially synthesized. For example, “human” domains may begenerated in rodents whose genome incorporates human immunoglobulingenes. See, e.g., Vaughan, et al, (1998), Nature Biotechnology, 16:535-539, e.g., to generate a fully human antibody. An antibody may bepolyclonal or monoclonal, though for purposes of the present inventionmonoclonal antibodies are generally preferred as therapeutic agents.Methods for generating antibodies that specifically bind to virtuallyany molecule of interest are known in the art. For example, monoclonalor polyclonal antibodies can be purified from natural sources, e.g.,from blood or ascites fluid of an animal that produces the antibody(e.g., following immunization with the molecule or an antigenic fragmentthereof) or can be produced recombinantly, in cell culture and, e.g.,purified from culture medium. Affinity purification may be used, e.g.,protein A/G affinity purification and/or affinity purification using theantigen as an affinity reagent. Suitable antibodies can be identifiedusing phage display and related techniques. See, e.g., Kaser, M. andHoward, G., “Making and Using Antibodies: A Practical Handbook” andSidhu, S., “Phage Display in Biotechnology and Drug Discovery”, CRCPress, Taylor and Francis Group, 2005, for further information. Methodsfor generating antibody fragments are well known. For example, F(ab′)₂fragments can be generated, for example, through the use of anImmunopure F(ab′)₂ Preparation Kit (Pierce) in which the antibodies aredigested using immobilized pepsin and purified over an immobilizedProtein A column. The digestion conditions (such as temperature andduration) may be optimized by one of ordinary skill in the art to obtaina good yield of F(ab′)₂. The yield of F(ab′)₂ resulting from thedigestion can be monitored by standard protein gel electrophoresis.F(ab′) can be obtained by papain digestion of antibodies, or by reducingthe S—S bond in the F(ab′)₂. As used herein, a “single-chain Fv” or“scFv” antibody fragment comprises the V_(H) and V_(L) domains of anantibody, wherein these domains are present in a single polypeptidechain. Typically, a scFv antibody further comprises a polypeptide linkerbetween the V_(H) and V_(L) domains, although other linkers could beused to connect the domains in certain embodiments.

The terms “approximately” or “about” in reference to a number generallyinclude numbers that fall within ±10%, in some embodiments ±5%, in someembodiments ±1%, in some embodiments ±0.5% of the number unlessotherwise stated or otherwise evident from the context (except wheresuch number would impermissibly exceed 100% of a possible value).

“Complement activation capacity” refers to the level of complementactivation that would result from exposure to a stimulus that causesmaximum complement activation. Typically, complement activation capacityis assessed using a sample obtained from a subject (e.g., a blood,plasma, serum, or other fluid sample, which may be dilutedappropriately), which sample may be exposed in vitro to a complementactivating stimulus. A heat-inactivated sample can be used as a control.It will be understood that the stimulus need not be sufficient to causemaximum complement activation in order to provide a measurement ofcomplement activation capacity. For example, the extent to whichcomplement activation occurs within a defined time period can provide anindication of complement activation capacity. Complement activation maybe measured using, e.g., a suitable assay such as a functional assaybased on hemolysis (e.g., lysis of sheep or chicken red blood cells);deposition or capture of complement activation products (e.g., C3a, C3b,iC3b, C5a, MAC), etc. Pathway-specific complement activation capacitymay be assessed using, e.g., appropriate stimuli and assay conditions(e.g., presence or absence of calcium ions in the assay composition) toactivate one or more than one of the pathways. For example, antibody(e.g., IgM or immune complexe) can be used to activate the classicalpathway; lipopolysaccharide (LPS) can be used to activate thealternative pathway, mannan can beused to activate the mannose-bindinglectin portion of the lectin pathway, etc. In some embodiments, thetotal classical complement activity in a sample is measured using a CH50test using antibody-sensitized sheep or chicken erythrocytes as theactivator of the classical complement pathway and various dilutions ofthe test sample to determine the amount required to give 50% lysis. Thepercent hemolysis can be determined spectrophotometrically. The higherthe dilution of the sample that can still achieve 50% lysis (i.e., themore diluted the sample), the greater complement activation capacity. Insome embodiments, an ELISA-based assay is used. In some embodiments,complement activation is assessed based on iC3b levels, e.g.,substantially as described in PCT/US2010/035871 (WO2010135717) (seeExamples). In some embodiments, complement activation is assessed basedon C3b levels, substantially as described in PCT/US2008/001483(WO/2008/097525) Examples 1 and 2, respectively. In some embodiments,complement activation via the classical pathway is assessed using theMicroVue CH50 Eq EIA Kit (classical pathway), MicroVue Bb Plus EIA Kit(alternative pathway), MicroVue iC3b EIA Kit, or MicroVue C3a Plus EIAKit (all from Quidel Corp.). In some embodiments, the amount of acomplement activation product is normalized to the amount of intact C3present in the sample prior to exposure to a complement activationstimulus.

A “complement component” or “complement protein” is a protein that isinvolved in activation of the complement system or participates in oneor more complement-mediated activities. Components of the classicalcomplement pathway include, e.g., C1q, C1r, C1s, C2, C3, C4, C5, C6, C7,C8, C9, and the C5b-9 complex, also referred to as the membrane attackcomplex (MAC) and active fragments or enzymatic cleavage products of anyof the foregoing (e.g., C3a, C3b, C4a, C4b, C5a, etc.). Components ofthe alternative pathway include, e.g., factors B, D, and properdin.Components of the lectin pathway include, e.g., MBL2, MASP-1, andMASP-2. Complement components also include cell-bound receptors forsoluble complement components, wherein such receptor mediates one ormore biological activities of such soluble complement componentfollowing binding of the soluble complement component. Such receptorsinclude, e.g., C5a receptor (C5aR), C3a receptor (C3aR), ComplementReceptor 1 (CR1), Complement Receptor 2 (CR2), Complement Receptor 3(CR3, also known as CD45), etc. It will be appreciated that the term“complement component” is not intended to include those molecules andmolecular structures that serve as “triggers” for complement activation,e.g., antigen-antibody complexes, foreign structures found on microbialor artificial surfaces, etc.

A “complement regulatory protein” is a protein involved in regulatingcomplement activity. A complement regulatory protein may down-regulatecomplement activity by, e.g., inhibiting complement activation or byinactivating or accelerating decay of one or more activated complementproteins. Examples of complement regulatory proteins include C1inhibitor, C4 binding protein, clusterin, vitronectin, CFH, factor I,and the cell-bound proteins CD46, CD55, CD59, CR1, CR2, and CR3.

“Linked”, as used herein with respect to two or more moieties, meansthat the moieities are physically associated or connected with oneanother to form a molecular structure that is sufficiently stable sothat the moieties remain associated under the conditions in which thelinkage is formed and, preferably, under the conditions in which the newmolecular structure is used, e.g., physiological conditions. In certainpreferred embodiments of the invention the linkage is a covalentlinkage. In other embodiments the linkage is noncovalent. Moieties maybe linked either directly or indirectly. When two moieties are directlylinked, they are either covalently bonded to one another or are insufficiently close proximity such that intermolecular forces between thetwo moieties maintain their association. When two moieties areindirectly linked, they are each linked either covalently ornoncovalently to a third moiety, which maintains the association betweenthe two moieties. In general, when two moieties are referred to as beinglinked by a “linking moiety” or “linking portion”, the linkage betweenthe two linked moieties is indirect, and typically each of the linkedmoieties is covalently bonded to the linking moiety. Two moieties may belinked using a “linker”. A linker can be any suitable moiety that reactswith the entities to be linked within a reasonable period of time, underconditions consistent with stability of the entities (portions of whichmay be protected as appropriate, depending upon the conditions), and insufficient amount, to produce a reasonable yield. Typically the linkerwill contain at least two functional groups, one of which reacts with afirst entity and the other of which reacts with a second entity. It willbe appreciated that after the linker has reacted with the entities to belinked, the term “linker” may refer to the part of the resultingstructure that originated from the linker, or at least the portion thatdoes not include the reacted functional groups. A linking moiety maycomprise a portion that does not participate in a bond with the entitiesbeing linked, and whose main purpose may be to spatially separate theentities from each other. Such portion may be referred to as a “spacer”.

“Polypeptide”, as used herein, refers to a polymer of amino acids,optionally including one or more amino acid analogs. A protein is amolecule composed of one or more polypeptides. A peptide is a relativelyshort polypeptide, typically between about 2 and 60 amino acids inlength, e.g., between 8 and 40 amino acids in length. The terms“protein”, “polypeptide”, and “peptide” may be used interchangeably.Polypeptides used herein may contain amino acids such as those that arenaturally found in proteins, amino acids that are not naturally found inproteins, and/or amino acid analogs that are not amino acids. As usedherein, an “analog” of an amino acid may be a different amino acid thatstructurally resembles the amino acid or a compound other than an aminoacid that structurally resembles the amino acid. A large number ofart-recognized analogs of the 20 amino acids commonly found in proteins(the “standard” amino acids) are known. One or more of the amino acidsin a polypeptide may be modified, for example, by the addition of achemical entity such as a carbohydrate group, a phosphate group, afarnesyl group, an isofarnesyl group, a fatty acid group, a linker forconjugation, functionalization, or other modification, etc. Certainnon-limiting suitable analogs and modifications are described inWO2004026328 and/or below. The polypeptide may be acetylated, e.g., atthe N-terminus and/or amidated, e.g., at the C-terminus.

In general, polypeptides may be obtained or produced using any suitablemethod known in the art. For example, polypeptides may be isolated fromnatural sources, produced in vitro or in vivo using recombinant DNAtechnology in suitable expression systems (e.g., by recombinant hostcells or transgenic non-human animals or plants), synthesized throughchemical means such as solid phase peptide synthesis and/or usingmethods involving chemical ligation of synthesized peptides (see, e.g.,Kent, S., J Pept Sci., 9(9):574-93, 2003 and U.S. Pub. No. 20040115774),or a combination of these. One of ordinary skill in the art wouldreadily select appropriate method(s). A polypeptide may comprise a tag,e.g., an epitope tag, which tag may facilitate purification and/ordetection of the polypeptide. Exemplary tags include, e.g., 6Xhis (SEQID NO: 70), HA, Myc, SNUT, FLAG, TAP, etc. In some embodiments, a tag iscleavable, e.g., the tag comprises a recognition site for cleavage by aprotease, or is separated from a portion complement inhibiting portionof the polypeptide by a linking portion that comprises a recognitionsite for cleavage by a protease. For example, a TEV protease cleavagesite can be used.

“Poxvirus” refers to a family of complex, double-stranded DNA virusesconstituting the family Poxyiridae. The family includes theorthopoxviruses, a genus of the family Poxyiridae, subfamilyChordopoxyirinae, comprising many species infecting mammals, includinghuman beings. Poxviruses are described in Fields, B N, et al., FieldsVirology, 3^(rd) ed., Lippincott Williams & Wilkins, 2001.Orthopoxviruses include, but are not limited to, vaccinia virus, variolavirus major, variola virus minor, cowpox virus, monkeypox virus,camelpox virus, swinepox virus, and ectromelia virus.

“Poxvirus complement control protein” refers to members of a family ofhomologous proteins encoded by a number of different poxviruses thatbind to one or more complement pathway proteins and inhibit either theclassical pathway of complement activation, the alternative pathway ofcomplement activation, the lectin pathway, or any combination of these.Poxvirus complement control proteins are members of the complementcontrol protein (CCP), also called regulators of complement activation(RCA) superfamily (Reid, K B M and Day, A J, Immunol Today, 10:177-80,1989).

“Recombinant host cells”, “host cells”, and other such terms, denoteprokaryotic or eukaryotic cells or cell lines that contain an exogenousnucleic acid (typically DNA) such as an expression vector comprising anucleic acid that encodes a polypeptide of interest. It will beunderstood that such terms include the descendants of the originalcell(s) into which the vector or other nucleic acid has been introduced.Appropriate host cells include any of those routinely used in the artfor expressing polynucleotides (e.g., for purposes of producingpolypeptide(s) encoded by such polynucleotides) including, for example,prokaryotes, such as E. coli; and eukaryotes, including for example,fungi, such as yeast (e.g., Pichia pastoris); insect cells (e.g., Sf9),plant cells, and animal cells, e.g., mammalian cells such as CHO, R1.1,B-W, L-M, African Green Monkey Kidney cells (e.g. COS-1, COS-7, BSC-1,BSC-40 and BMT-10) and cultured human cells. The exogenous nucleic acidmay be stably maintained as an episome such as a plasmid or may at leastin part be integrated into the host cell's genome, optionally afterbeing copied or reverse transcribed. Terms such as “host cells”, etc.,are also used to refer to cells or cell lines that can be used asrecipients for an exogenous nucleic acid, prior to introduction of thenucleic acid. A “recombinant polynucleotide” is a polynucleotide thatcontains nucleic acid sequences that are not found joined directly toone another in nature. For example, the nucleic acid sequences may occurin different genes or different species or one or more of thesequence(s) may be a variant of a naturally occurring sequence or may atleast in part be an artificial sequence that is not homologous to anaturally occurring sequence. A “recombinant polypeptide” is apolypeptide that is produced by transcription and translation of anexogenous nucleic acid by a recombinant host cell or by a cell-free invitro expression system and/or that contains amino acid sequences thatare not found joined directly to one another in nature. In the lattercase, the recombinant polypeptide may be referred to as a “chimericpolypeptide”. The amino acid sequences in a chimeric polypeptide may,for example, occur in different genes or in different species or one ormore of the sequence(s) may be a variant of a naturally occurringsequence or may at least in part be an artificial sequence that is nothomologous to a naturally occurring sequence. It will be understood thata chimeric polypeptide may comprise two or more polypeptide. Forexample, first and second polypeptides A and B of a chimeric polypeptidemay be directly linked (A-B or B-A) or may be separated by a thirdpolypeptide portion C (A-C-B or B-C-A). In some embodiments, portion Crepresents a polypeptide linker which may, for example, comprisemultiple glycine and/or serine residues. In some embodiments, two ormore polypeptides may be linked by non-polypeptide linker(s).

“Reactive functional groups” as used herein refers to groups including,but not limited to, olefins, acetylenes, alcohols, phenols, ethers,oxides, halides, aldehydes, ketones, carboxylic acids, esters, amides,cyanates, isocyanates, thiocyanates, isothiocyanates, amines,hydrazines, hydrazones, hydrazides, diazo, diazonium, nitro, nitriles,mercaptans, sulfides, disulfides, sulfoxides, sulfones, sulfonic acids,sulfinic acids, acetals, ketals, anhydrides, sulfates, sulfenic acidsisonitriles, amidines, imides, imidates, nitrones, hydroxylamines,oximes, hydroxamic acids thiohydroxamic acids, allenes, ortho esters,sulfites, enamines, ynamines, ureas, pseudoureas, semicarbazides,carbodiimides, carbamates, imines, azides, azo compounds, azoxycompounds, and nitroso compounds, N-hydroxysuccinimide esters,maleimides, sulfhydryls, and the like. Methods to prepare each of thesefunctional groups are well known in the art and their application to ormodification for a particular purpose is within the ability of one ofskill in the art (see, for example, Sandler and Karo, eds. ORGANICFUNCTIONAL GROUP PREPARATIONS, Academic Press, San Diego, 1989, andHermanson, G., Bioconjugate Techniques, 2^(nd) ed., Academic Press, SanDiego, 2008).

“Specific binding” generally refers to a physical association between atarget polypeptide (or, more generally, a target molecule) and a bindingmolecule such as an antibody or ligand. The association is typicallydependent upon the presence of a particular structural feature of thetarget such as an antigenic determinant, epitope, binding pocket orcleft, recognized by the binding molecule. For example, if an antibodyis specific for epitope A, the presence of a polypeptide containingepitope A or the presence of free unlabeled A in a reaction containingboth free labeled A and the binding molecule that binds thereto, willreduce the amount of labeled A that binds to the binding molecule. It isto be understood that specificity need not be absolute but generallyrefers to the context in which the binding occurs. For example, it iswell known in the art that numerous antibodies cross-react with otherepitopes in addition to those present in the target molecule. Suchcross-reactivity may be acceptable depending upon the application forwhich the antibody is to be used. One of ordinary skill in the art willbe able to select antibodies or ligands having a sufficient degree ofspecificity to perform appropriately in any given application (e.g., fordetection of a target molecule, for therapeutic purposes, etc). It isalso to be understood that specificity may be evaluated in the contextof additional factors such as the affinity of the binding molecule forthe target versus the affinity of the binding molecule for othertargets, e.g., competitors. If a binding molecule exhibits a highaffinity for a target molecule that it is desired to detect and lowaffinity for nontarget molecules, the antibody will likely be anacceptable reagent. Once the specificity of a binding molecule isestablished in one or more contexts, it may be employed in other,preferably similar, contexts without necessarily re-evaluating itsspecificity. In some embodiments, the affinity (as measured by theequilibrium dissociation constant, Kd) of two molecules, e.g., twomolecules that exhibit specific binding, is 10⁻³ M or less, e.g., 10⁻⁴ Mor less, e.g., 10⁻⁵ M or less, e.g., 10⁻⁶M or less, 10⁻⁷M or less, 10⁻⁸Mor less, or 10⁻⁹ M or less under the conditions tested, e.g., underphysiological conditions (e.g., conditions such as salt concentration,pH, and/or temperature, etc., that reasonably approximate correspondingconditions in vivo), or other conditions of the assay. Binding affinitycan be measured using any of a variety of methods known in the art. Forexample, assays based on isothermal titration calorimetry or surfaceplasmon resonance (e.g., Biacore® assays) can be used in certainembodiments.

A “subject” treated according to the instant invention is typically ahuman, a non-human primate, or another mammal (e.g., a mouse or rat). Itwill be appreciated that, at least in embodiments wherein a complementinhibitor is administered, the subject should express at least onecomplement component that can be inhibited by the particular complementinhibitor used. For example, a complement inhibitor specific for primatecomplement would typically be administered to a human or non-humanprimate or an animal model that has been genetically engineered toexpress human complement component(s). In some embodiments the subjectis male. In some embodiments the subject is female. In some embodiments,a human subject is at least 12 years of age. In some embodiments asubject is an adult, e.g., a human at least 18 years of age, e.g.,between 18 and 100 years of age. In some embodiments a subject is atleast 40, 45, 50, 55, 60, 65, 70, 75, or 80 years of age. In someembodiments the subject is a child, e.g., a human between 0 and 4 yearsof age, or between 5 and 11 years of age.

“Treating”, as used herein in regard to treating a subject, refers toproviding treatment, i.e, providing any type of medical or surgicalmanagement of a subject. The treatment can be provided in order toreverse, alleviate, inhibit the progression of, prevent or reduce thelikelihood of a disease, or in order to reverse, alleviate, inhibit orprevent the progression of, prevent or reduce the likelihood of one ormore symptoms or manifestations of a disease. “Prevent” refers tocausing a disease or symptom or manifestation of a disease not to occurfor at least a period of time in at least some individuals, e.g.,individuals at risk of developing the disease, symptom, ormanifestation. Treating can include administering a compound orcomposition to the subject following the development of one or moresymptoms or manifestations indicative of a disease, e.g., in order toreverse, alleviate, reduce the severity of, and/or inhibit or preventthe progression of the disease and/or to reverse, alleviate, reduce theseverity of, and/or inhibit or one or more symptoms or manifestations ofthe disease. A compound or composition can be administered to a subjectwho has developed a disease, or is at increased risk of developing thedisease relative to a member of the general population, optionally amember who is matched with the subject in terms of age, sex, and/orother demographic variable(s).

A “variant” of a particular polypeptide or polynucleotide has one ormore alterations (e.g., additions, substitutions, and/or deletions,which may be referred to collectively as “mutations”) with respect tothe polypeptide or nucleic acid, which may be referred to as the“original polypeptide” or “original polynucleotide”, respectively. Thusa variant can be shorter or longer than the polypeptide orpolynucleotide of which it is a variant. The terms “variant” encompasses“fragments”. A “fragment” is a continuous portion of a polypeptide thatis shorter than the original polypeptide. In certain embodiments of theinvention a variant polypeptide has significant sequence identity to theoriginal polypeptide over a continuous portion of the variant thatcomprises at least 50%, preferably at least 60%, at least 70%, at least80%, at least 90%, at least 95%, or more, of the length of the variantor the length of the polypeptide, (whichever is shorter). In certainembodiments of the invention a variant polypeptide has substantialsequence identity to the original polypeptide over a continuous portionof the variant that comprises at least 50%, preferably at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, or more, of thelength of the variant or the length of the polypeptide, (whichever isshorter). In a non-limiting embodiment a variant has at least 80%identity to the original sequence over a continuous portion of thevariant that comprises between 90% and 100% of the variant, e.g., over100% of the length of the variant or the length of the polypeptide,(whichever is shorter). In another non-limiting embodiment a variant hasat least 80% identity to the original sequence over a continuous portionof the variant that comprises between 90% and 100% of the variant, e.g.,over 100% of the length of the variant or the length of the polypeptide,(whichever is shorter). In specific embodiments the sequence of avariant polypeptide has N amino acid differences with respect to anoriginal sequence, wherein N is any integer between 1 and 10. In otherspecific embodiments the sequence of a variant polypeptide has N aminoacid differences with respect to an original sequence, wherein N is anyinteger between 1 and 20. An amino acid “difference” refers to asubstitution, insertion, or deletion of an amino acid.

In certain embodiments of the invention a fragment or variant possessessufficient structural similarity to the original polypeptide so thatwhen its 3-dimensional structure (either actual or predicted structure)is superimposed on the structure of the original polypeptide, the volumeof overlap is at least 70%, preferably at least 80%, more preferably atleast 90% of the total volume of the structure of the originalpolypeptide. A partial or complete 3-dimensional structure of thefragment or variant may be determined by crystallizing the protein,which can be done using standard methods. Alternately, an NMR solutionstructure can be generated, also using standard methods. A modelingprogram such as MODELER (Sali, A. and Blundell, T L, J. Mol. Biol., 234,779-815, 1993), or any other modeling program, can be used to generate apredicted structure. If a structure or predicted structure of a relatedpolypeptide is available, the model can be based on that structure. ThePROSPECT-PSPP suite of programs can be used (Guo, J T, et al., NucleicAcids Res. 32(Web Server issue):W522-5, Jul. 1, 2004).

In many embodiments one, more than one, or all biological functions oractivities of a variant or fragment is substantially similar to that ofthe corresponding biological function or activity of the originalmolecule. In certain embodiments the activity of a variant or fragmentmay be at least 20%, at least 50%, at least 60%, at least 70%, at least80%, or at least 90% of the activity of the original molecule, up toapproximately 100%, approximately 125%, or approximately 150% of theactivity of the original molecule. In certain embodiments an activity ofa variant or fragment is such that the amount or concentration of thevariant needed to produce an effect is within 0.5 to 5-fold of theamount or concentration of the original molecule needed to produce thateffect. The invention contemplates use of variants of any of thecomplement inhibiting polypeptides disclosed herein, wherein the variantinhibits complement sufficiently to be useful in a method describedherein. In some embodiments, a variant lacks or has a substantiallyreduction in a property that may be undesired such as immunogenicity.

As used herein, “alkyl” refers to a saturated straight, branched, orcyclic hydrocarbon having from about 1 to about 22 carbon atoms (and allcombinations and subcombinations of ranges and specific numbers ofcarbon atoms therein), with from about 1 to about 12, or about 1 toabout 7 carbon atoms being preferred in certain embodiments of theinvention. Alkyl groups include, but are not limited to, methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n- pentyl, cyclopentyl,isopentyl, neopentyl, n-hexyl, isohexyl, cyclohexyl, cyclooctyl,adamantyl, 3-methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl.

As used herein, “halo” refers to F, Cl, Br or I.

As used herein, “alkanoyl” refers to an optionally substituted straightor branched aliphatic acyclic residue having about 1 to 10 carbon atoms(and all combinations and subcombinations of ranges and specific numberof carbon atoms) therein, e.g., from about 1 to 7 carbon atoms which, aswill be appreciated, is attached to a terminal C═O group with a singlebond (and may also be referred to as an “acyl group”). Alkanoyl groupsinclude, but are not limited to, formyl, acetyl, propionyl, butyryl,isobutyryl, pentanoyl, isopentanoyl, 2-methyl-butyryl,2,2-dimethoxypropionyl, hexanoyl, heptanoyl, octanoyl, and the like, andfor purposes of the present invention a formyl group is considered analkanoyl group. “Lower alkanoyl” refers to an optionally substitutedstraight or branched aliphatic acyclic residue having about 1 to about 5carbon atoms (and all combinations and subcombinations of ranges andspecific number of carbon atoms). Such groups include, but are notlimited to, formyl, acetyl, propionyl, butyryl, isobutyryl, pentanoyl,isopentanoyl, etc.

As used herein, “aryl” refers to an optionally substituted, mono- orbicyclic aromatic ring system having from about 5 to about 14 carbonatoms (and all combinations and subcombinations of ranges and specificnumbers of carbon atoms therein), with from about 6 to about 10 carbonsbeing preferred. Non-limiting examples include, for example, phenyl andnaphthyl.

As used herein, “aralkyl” refers to alkyl radicals bearing an arylsubstituent and having from about 6 to about 22 carbon atoms (and allcombinations and subcombinations of ranges and specific numbers ofcarbon atoms therein), with from about 6 to about 12 carbon atoms beingpreferred in certain embodiments. Aralkyl groups can be optionallysubstituted. Non-limiting examples include, for example, benzyl,naphthylmethyl, diphenylmethyl, triphenylmethyl, phenylethyl, anddiphenylethyl.

As used herein, the terms “alkoxy” and “alkoxyl” refer to an optionallysubstituted alkyl-O-group wherein alkyl is as previously defined.Exemplary alkoxy and alkoxyl groups include methoxy, ethoxy, n-propoxy,i-propoxy, n-butoxy, and heptoxy.

As used herein, “carboxy” refers to a —C(═O)OH group.

As used herein, “alkoxycarbonyl” refers to a —C(═O)O-alkyl group, wherealkyl is as previously defined.

As used herein, “aroyl” refers to a —C(═O)-aryl group, wherein aryl isas previously defined. Exemplary aroyl groups include benzoyl andnaphthoyl.

The term “cyclic ring system” refers to an aromatic or non-aromatic,partially unsaturated or fully saturated, 3- to 10-membered ring system,which includes single rings of 3 to 8 atoms in size and bi- andtri-cyclic ring systems which may include aromatic 5- or 6-membered arylor aromatic heterocyclic groups fused to a non-aromatic ring. Theseheterocyclic rings include those having from 1 to 3 heteroatomsindependently selected from the group consisting of oxygen, sulfur, andnitrogen. In certain embodiments, the term heterocyclic refers to anon-aromatic 5-, 6-, or 7-membered ring or a polycyclic group wherein atleast one ring atom is a heteroatom selected from the group consistingof O, S, and N, including, but not limited to, a bi- or tri-cyclicgroup, comprising fused six-membered rings having between one and threeheteroatoms independently selected from the group consisting of theoxygen, sulfur, and nitrogen. In some embodiments, “cyclic ring system”refers to a cycloalkyl group which, as used herein, refers to groupshaving 3 to 10, e.g., 4 to 7 carbon atoms. Cycloalkyls include, but arenot limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl and the like, which, is optionally substituted. In someembodiments, “cyclic ring system” refers to a cycloalkenyl orcycloalkynyl moiety, which is optionally substituted.

Typically, substituted chemical moieties include one or moresubstituents that replace hydrogen. Exemplary substituents include, forexample, halo, alkyl, cycloalkyl, aralkyl, aryl, sulfhydryl, hydroxyl(—OH), alkoxyl, cyano (—CN), carboxyl (—COOH), —C(═O)O-alkyl,aminocarbonyl (—C(═O)NH₂), —N-substituted aminocarbonyl (—C(═O)NHR″),CF₃, CF₂CF₃, and the like. In relation to the aforementionedsubstituents, each moiety R″ can be, independently, any of H, alkyl,cycloalkyl, aryl, or aralkyl, for example.

As used herein, “L-amino acid” refers to any of the naturally occurringlevorotatory alpha-amino acids normally present in proteins or the alkylesters of those alpha-amino acids. The term “D-amino acid” refers todextrorotatory alpha-amino acids. Unless specified otherwise, all aminoacids referred to herein are L-amino acids.

As used herein, an “aromatic amino acid” is an amino acid that comprisesat least one aromatic ring, e.g., it comprises an aryl group.

As used herein, an “aromatic amino acid analog” is an amino acid analogthat comprises at least one aromatic ring, e.g., it comprises an arylgroup.

II. Methods of Treating Disorders Using Complement Inhibitors

The present invention provides, among other things, methods of treatingchronic complement-mediated disorders using complement inhibitors. Forexample, the invention provides methods of treating chronic respiratorysystem disorders using complement inhibitors. In some aspects, theinventive methods are based at least in part on the recognition thatcomplement inhibitors have a prolonged duration of action in treating avariety of disorders, e.g., chronic respiratory disorders, as compared,for example, with their plasma half-life and/or their duration of actionfor inhibiting plasma complement activation capacity. In some aspects,the invention provides methods of treating a chronic complement-mediateddisorder by administering multiple doses of a complement inhibitor,wherein the complement inhibitor is administered according to a dosingschedule that utilizes the prolonged effect of complement inhibition.

As used herein, a “chronic disorder” is a disorder that persists for atleast 3 months and/or is accepted in the art as being a chronicdisorder. In many embodiments, a chronic disorder persists for at least6 months, e.g., at least 1 year, or more, e.g., indefinitely. One ofordinary skill in the art will appreciate that at least somemanifestations of various chronic disorders may be intermittent and/ormay wax and wane in severity over time. A chronic disorder may beprogressive, e.g., having a tendency to become more severe or affectlarger areas over time. A number of chronic complement-mediateddisorders are discussed herein. Varioue embodiments of the inventionpertaining to chronic, complement-mediated respiratory disorders, inparticular asthma and COPD, are discussed in most detail herein, but itshould be understood that the various aspects of the invention encompassembodiments pertaining to any chronic complement-mediated disorderincluding, but not limited to, the specific disorders disclosed herein.Accordingly, where an embodiment herein refers to a chronic respiratorydisorder, the invention provides analogous embodiments pertaining toother complement-mediated disorders, e.g., chronic disorders in whichcomplement activation (e.g., excessive or inappropriate complementactivation) is involved, e.g., as a contributing and/or at leastpartially causative factor. For convenience, disorders are sometimesgrouped by reference to an organ or system that is often particularlyaffected in subjects suffering from the disorder. It will be appreciatedthat a number of disorders can affect multiple organs or systems, andthe classification herein is in no way limiting. Furthermore, a numberof manifestations (e.g., symptoms) may occur in subjects suffering fromany of a number of different disorders. In some aspects, the inventionprovides methods of treating a subject in need of treatment for suchmanifestation(s), e.g., methods for alleviating such manifestation(s),the methods comprising administering a complement inhibitor to thesubject according to an inventive dosing schedule (e.g., a dosingschedule that employs an inventive dosing interval). In someembodiments, a subject suffers from multiple complement-mediateddisorders. Non-limiting information regarding disorders of interestherein may be found, e.g., in standard textbooks of internal medicinesuch as Cecil Textbook of Medicine (e.g., 23rd edition), Harrison'sPrinciples of Internal Medicine (e.g., 17th edition), and/or standardtextbooks focusing on particular areas of medicine, particular bodysystems or organs, and/or particular disorders.

In some embodiments, a chronic complement-mediated disorder is aTh2-associated disorder. As used herein, a Th2-associated disorder is adisorder characterized by an excessive number and/or excessive orinappropriate activity of CD4+ helper T cells of the Th2 subtype (“Th2cells”) in the body or a portion thereof, e.g., in at least one tissue,organ, or structure. For example, there may be a predominance of Th2cells relative to CD4+ helper T cells of the Th1 subtype (“Th1 cells”)e.g., in at least one tissue, organ, or structure affected by adisorder. As known in the art, Th2 cells typically secretecharacteristic cytokines such as interleukin-4 (IL-4), interleukin-5(IL-5), and interleukin-13 (IL-13), while Th1 cells typically secreteinterferon-γ (IFN-γ) and tumor necrosis factor β (TNF β). In someembodiments, a Th2-associated disorder is characterized by excessiveproduction and/or amount of IL-4, IL-5, and/or IL-13, e.g., relative toIFN-γ and/or TNF β e.g., in at least some at least one tissue, organ, orstructure.

In some embodiments, a chronic complement-mediated disorder is aTh17-associated disorder. As used herein, a Th17-associated disorder isa disorder characterized by an excessive number and/or excessive orinappropriate activity of CD4+ helper T cells of the Th17 subtype (“Th17cells”) in the body or a portion thereof, e.g., in at least one tissue,organ, or structure. For example, there may be a predominance of Th17cells relative to Th1 and/or Th2 cells, e.g., in at least one tissue,organ, or structure affected by a disorder. In some embodiments apredominance of Th17 cells is a relative predominance, e.g., the ratioof Th17 cells to Th1 cells and/or the ratio of Th17 cells to Th2 cells,is increased relative to normal values. In some embodiments the ratio ofTh17 cells to T regulatory cells (CD4⁺CD25⁺ regulatory T cells, alsotermed “Treg cells”), is increased relative to normal values. Formationof Th17 cells and/or activation of Th 17 cells is promoted by variouscytokines, e.g., interleukin 6 (IL-6), interleukin 21 (IL-21),interleukin 23 (IL-23), and/or interleukin 1β (IL-1β). Formation of Th17cells encompasses differentiation of precursor T cells, e.g., naïve CD4+T cells, towards a Th17 phenotype and their maturation into functionalTh17 cells. In some embodiments, formation of Th17 cells encompasses anyaspect of development, proliferation (expansion), survival, and/ormaturation of Th17 cells. In some embodiments, a Th17-associateddisorder is characterized by excessive production and/or amount of IL-6,IL-21, IL-23, and/or IL-1β. Th17 cells typically secrete characteristiccytokines such as interleukin-17A (IL-17A), interleukin-17F (IL-17F),interleukin-21 (IL-21), and interleukin-22 (IL-22). In some embodiments,a Th17-associated disorder is characterized by excessive productionand/or amount of a Th17 effector cytokine, e.g., IL-17A, IL-17F, IL-21,and/or IL-22. In some embodiments excessive production or amount of acytokine is detectable in the blood. In some embodiments excessiveproduction or amount of a cytokine is detectable locally, e.g., in atleast one tissue, organ or structure. In some embodiments aTh17-associated disorder is associated with a decreased number of Tregsand/or decreased amount of a Treg-associated cytokine. In someembodiments a Th17 disorder is any chronic inflammatory disease, whichterm encompasses a range of ailments characterized by self-perpetuatingimmune insults to a variety of tissues and that seem to be dissociatedfrom the initial insult that caused the ailment (which may be unknown).In some embodiments a Th17-associated disorder is any autoimmunedisease. Many if not most “chronic inflammatory diseases” may in fact beauto-immune diseases. Examples of Th17-associated disorders includeinflammatory skin diseases such as psoriasis and atopic dermatitis;systemic scleroderma and sclerosis; inflammatory bowel disease (IBD)(such as Crohn's disease and ulcerative colitis); Behcet's Disease;dermatomyositis; polymyositis; multiple sclerosis (MS); dermatitis;meningitis; encephalitis; uveitis; osteoarthritis; lupus nephritis;rheumatoid arthritis (RA), Sjorgen's syndrome, multiple sclerosis,vasculitis; central nervous system (CNS) inflammatory disorders, chronichepatitis; chronic pancreatitis, glomerulonephritis; sarcoidosis;thyroiditis, pathologic immune responses to tissue/organ transplantation(e.g., transplant rejection); COPD, asthma, bronchiolitis,hypersensitivity pneumonitis, idiopathic pulmonary fibrosis (IPF),periodontitis, and gingivitis. In some embodiments a Th17 disease is aclassically known auto-immmune disease such as Type I diabetes orpsoriasis. In some embodiments a Th17-associated disorder is age-relatedmacular degeneration.

In some aspects, the present disclosure provides the insight thatcomplement activation and Th17 cells participate in a cycle thatinvolves dendritic cells and antibodies and that contributes tomaintenance of a pathologic immunologic microenvironment underlying arange of disorders. Without wishing to be bound by any theory, thepathologic immunologic microenvironment, once established, isself-sustaining and contributes to cell and tissue injury. Dendriticcells (DCs) are a type of white blood cell that occur in most tissues ofthe body, particularly those exposed to the external environment, suchas skin and mucosal surfaces, and in the blood (where they may be foundin an immature state). Immature DCs sample the surrounding environmentfor pathogens through, e.g., pattern recognition receptors such astoll-like receptors (TLRs). In response to various stimuli (e.g.,pathogen-associated substances or other danger signals, inflammatorycytokines, and/or antigen-activated T cells), DCs mature and migrate tolymphoid tissues, where they act as antigen-presenting cells andactivate other immune system cells, such as T cells and B cells, bypresenting them with antigen fragments together with non-antigenspecific costimulatory molecules. DC stimulation promotes Th cellproliferation, activation, and differentiation into effector Th cells.Effector Th cells “help” cytotoxic T cells, B cells, and macrophages by,e.g., secreting cytokines that have various stimulatory effects. Th helpcan, for example, enhance proliferation and activation of cytotoxic Tcells, stimulate B cell proliferation and maturation and antibodyproduction. Of particular importance in accordance with certain aspectsof the present disclosure, mature DCs are capable of causing CD4+ helperT cells to differentiate into Th17 cells, which in turn stimulatematuration and activation of B cells, resulting in production ofantibodies.

The antibody response is generally polyclonal, with most antibodiesbeing of low affinity. However, certain of these antibodies may becross-reactive with self proteins, such as self proteins that have beenenzymatically or non-enzymatically chemically modified in the bodypost-translationally in any of a variety of ways. Such self proteinsmay, for example, be exposed at the surface of cells, present in theinterstitial space, and/or circulating in the blood. Modifications ofself proteins may include, e.g., acylation and/or glycation(non-enzymatic formation of a covalent bond between a protein or lipidand a sugar). For example, proteins can be oxidized in numerous ways,which can be classified into at least three categories. A firstmechanism involves oxidative cleavages in either the protein backbone oramino acid side chains, e.g., side chains of Pro, Arg, Lys, Thr, Glu orAsp residues, which may occur by direct oxidation with reactive oxygenspecies (ROS). Certain ROS are produced during normal cellularmetabolism, and various mechanisms exist to defend against thepotentially damaging effects of such compounds. Examples of ROS include,e.g., superoxide anion, hydrogen peroxide, and peroxynitrite. Excessivelevels of reactive oxygen species (ROS) can result from the environmentand/or defects in cellular processes or antioxidant mechanisms,resulting in high levels of oxidative stress. A second mechanism ofprotein oxidation is by addition of lipid oxidation products such as4-hydroxy-2-noneal, 2-propenal or malondialdehyde to proteins. In athird mechanism, carbonyl groups are generated in proteins by oxidationof advanced glycation end (AGE) products. AGEs can form as a result of achain of chemical reactions after an initial glycation reaction.Examples of AGE-modified sites are carboxymethyllysine (CML) andcarboxyethyllysine (CEL). ROS can degrade polyunsaturated lipids,forming malondialdehyde, a reactive aldehyde that forms covalent proteinadducts referred to as advanced lipoxidation end-products (ALEs).Carboxyethylpyrrole (CEP) protein modifications are generated fromoxidation of docosahexaenoate-containing lipids.

Modified self proteins (e.g., malondialdehyde-modified proteins,CEP-modified proteins) may contain epitopes recognized as non-self bythe immune system, e.g., by antibodies. Binding of antibodies to selfproteins leads to complement activation, e.g., via the classicalpathway. Once initiated, classical pathway-mediated complementactivation is amplified by the alternative pathway. In accordance withcertain aspects of the present disclosure, activated complementpolarizes DCs to sustain the Th17 phenotype. For example, DCs may bepolarized towards secretion of cytokines such as IL-23 that promote Th17formation and/or activation. Complement cleavage products such as theanaphylotoxins (e.g., C3a, C4a, and/or C5a) and/or products of C3cleavage and degradation such as iC3b or C3d may bind to DC cell surfacereceptors and contribute towards polarizing DCs to sustain the Th17phenotype. An example of how complement can polarize DCs is theactivation of dendritic cells by aluminum oxide. Aluminum oxide iswidely used as an adjuvant to vaccines. Aluminum oxide activatescomplement and this stimulates DCs into promoting and sustaining Th2 andTh17 phenotypes. Complement can polarize other types ofantigen-presenting cells as well. Monocytes and macrophages can act asantigen-presenting cells and can similarly be polarized by complementactivation. In some aspects, the cycle may be summarized as follows: (1)Mature dendritic cells in an environment of high complement activationstimulate Th17 cell phenotypic differentiation; (2) Th17 T cellsstimulate polyclonal B-cell expansion, leading to the production ofpolyclonal, self-reactive antibodies against, e.g., modified selfproteins, such as carbonyl-modified self-proteins; (3) Carbonyl-modifiedself-proteins can be generated as a result of oxidative stress. This canarise, for example, from pollutants, cigarette smoke, or allergens; (4)Self-reactive antibodies against carbonyl-modified self-proteins helppromote or sustain an environment of high complement activation; (5)High complement activation drives antigen-presenting cells intosustaining a Th17 micro-environment.

The effector pathways that lead this cycle to inflict tissue damage canbe varied, but, without wishing to be bound by any theory, it isbelieved that a principal pathway is via macrophages. In some aspects,IL-17 secreted by Th17 cells, itself or in combination with one or moreother cytokines such as interferon gamma (IFN-γ) contributes tomacrophage activation and/or polarization towards an M1 phenotype.M1-polarized macrophages are immune effector cells that arecharacterized by expression of high levels of proinflammatory cytokines,high production of reactive nitrogen and oxygen intermediates, and mayexhibit strong cytotoxic activity against targets such as microbes andtumor cells. Macrophages, e.g., M1-polarized macrophages, and theproducts they produce can lead to tissue damage and are importantmediators of immunopathology. Modification of self proteins and othercellular components by reactive nitrogen and oxygen species can renderthem dysfunctional, thereby interfering with normal cellular processes.Dysfunctional modified proteins can accumulate to toxic levels, whichcan lead to cell death. Macrophages are also capable of direct killingof altered self cells, e.g., self cells that have oxidatively modifiedproteins or lipids exposed at their cell surface. Reactive nitrogen andoxygen species produced by macrophages can amplify oxidative stress,resulting in further modification of self proteins by mechanisms such asthose described above, which produces new targets for self-reactiveantibodies and macrophages. The antibodies further activate complement,which maintains DC polarization towards a Th17-promoting phenotype.Thus, a vicious cycle is perpetuated in which Th17 cells activate Bcells, resulting in polyclonal antibody production and consequentcomplement activation, which in term promotes DC polarization towards aTh17-promoting phenotype that drives continued stimulation of B cellsand antibody production. For purposes hereof, this cycle, alsosummarized above, may be referred to as the “dendritic cell—Th 17 cell—Bcell-antibody-complement-dendritic cell” cycle, abbreviated asDC-Th17-B-Ab-C-DC cycle. Polarization of macrophages to an M1 phenotypeand production of ROS that can directly damage cellular components mayoccur as “outputs” of this feedback loop. The pathologic consequencesthat result from DC-Th17-B-Ab-C-DC cycle and its outputs may vary indifferent tissues or organs. For example, in the respiratory system,they may at least in part underlie chronic respiratory diseases such asasthma and COPD. In the eye, they may at least in part underlie chronicdisorders such as age-related macular degeneration. In the skin, theymay at least in part underlie psoriasis. In the pancreas, they may atleast in part underlie Type I diabetes.

In some embodiments, a chronic complement-mediated disorder is anIgE-associated disorder. As used herein, an “IgE-associated disorder” isa disorder characterized by excessive and/or inappropriate productionand/or amount of IgE, excessive or inappropriate activity of IgEproducing cells (e.g., IgE producing B cells or plasma cells), and/orexcessive and/or inappropriate activity of IgE responsive cells such aseosinophils or mast cells. In some embodiments, an IgE-associateddisorder is characterized by elevated levels of total IgE and/or in someembodiments, allergen-specific IgE, in the plasma of a subject and/orlocally.

In some embodiments, a chronic complement-mediated disorder ischaracterized by complement-mediated hemolysis, e.g.,complement-mediated hemolysis attributable to deficiency or mutation ofone or more endogenous complement regulatory proteins. In someembodiments, a chronic complement-mediated disorder is not characterizedby hemolysis attributable, e.g., to deficiency or mutation of one ormore endogenous complement regulatory proteins.

In some embodiments, a chronic complement-mediated disorder ischaracterized by the presence of autoantibodies and/or immune complexesin the body, which may activate complement via, e.g., the classicalpathway. Autoantibodies may, for example, bind to self antigens, e.g.,on cells or tissues in the body. In some embodiments, autoantibodiesbind to antigens in blood vessels, skin, nerves, muscle, connectivetissue, heart, kidney, thyroid, etc. In some embodiments, a chroniccomplement-mediated disorder is not characterized by autoantibodiesand/or immune complexes.

In some embodiments, the invention provides methods for treating achronic complement-mediated disorder by administering multiple doses ofa complement inhibitor, wherein the complement inhibitor is administeredaccording to a dosing schedule that utilizes the prolonged effect ofcomplement inhibition. “Dosing schedule” refers to the timing ofadministration of a compound (or composition containing a compound). Insome embodiments, an inventive method utilizes an increased dosinginterval as compared, for example, with a dosing interval that aims tomaintain a significant level of complement inhibitor and/or asignificant level of complement inhibition in the body substantiallythroughout a treatment period. In some embodiments, an inventive methodutilizes an increased dosing interval as compared, for example, with adosing interval that aims to expose tissue(s) or organ(s) affected by acomplement-mediated disorder to a significant level of complementinhibitor and/or maintain a significant level of complement inhibitionin such tissue(s) or organ(s) (and/or in body fluids contacting orwithin such tissue(s) or organ(s)) substantially throughout a treatmentperiod. As used herein, “dosing interval” refers to the time intervalbetween administration of successive doses of a compound (or compositioncomprising a compound).

In some embodiments, a chronic complement-mediated disorder is arespiratory disorder. In some embodiments, a chronic respiratorydisorder is asthma or chronic obstructive pulmonary disease (COPD). Insome embodiments, a chronic respiratory disorder is pulmonary fibrosis(e.g., idiopathic pulmonary fibrosis), radiation-induced lung injury,allergic bronchopulmonary aspergillosis, hypersensitivity pneumonitis(also known as allergic alveolitis), eosinophilic pneumonia,interstitial pneumonia, sarcoid, Wegener's granulomatosis, orbronchiolitis obliterans.

In some embodiments, a chronic complement-mediated disorder is allergicrhinitis, rhinosinusitis, or nasal polyposis. In some embodiments, theinvention provides a method of treating a subject in need of treatmentfor allergic rhinitis, rhinosinusitis, or nasal polyposis, the methodcomprising administering a complement inhibitor according to a dosingschedule described herein to a subject in need of treatment for thedisorder.

In some embodiments, a chronic complement-mediated disorder is adisorder that affects the musculoskeletal system. Examples of suchdisorders include inflammatory joint conditions (e.g., arthritis such asrheumatoid arthritis or psoriatic arthritis, juvenile chronic arthritis,spondyloarthropathies Reiter's syndrome, gout). In some embodiments, amusculoskeletal system disorder results in symptoms such as pain,stiffness and/or limitation of motion of the affected body part(s).Inflammatory myopathies include dermatomyositis, polymyositis, andvarious others are disorders of chronic muscle inflammation of unknownetiology that result in muscle weakness. In some embodiments, a chroniccomplement-mediated disorder is myasthenia gravis. In some embodiments,the invention provides a method of treating any of the foregoingdisorders affecting the musculoskeletal system, the method comprisingadministering a complement inhibitor according to a dosing scheduledescribed herein to a subject in need of treatment for the disorder.

In some embodiments, a chronic complement-mediated disorder is adisorder that affects the integumentary system. Examples of suchdisorders include, e.g., atopic dermatitis, psoriasis, pemphigus,systemic lupus erythematosus, dermatomyositis, scleroderma,sclerodermatomyositis, Sjögren syndrome, and chronic urticaria. In someaspects, the invention provides a method of treating any of theforegoing disorders affecting the integumentary system, the methodcomprising administering a complement inhibitor according to a dosingschedule described herein to a subject in need of treatment for thedisorder.

In some embodiments, a chronic complement-mediated disorder affects thenervous system, e.g., the central nervous system (CNS) and/or peripheralnervous system (PNS). Examples of such disorders include, e.g., multiplesclerosis, other chronic demyelinating diseases, amyotrophic lateralsclerosis, chronic pain, stroke, allergic neuritis, Huntington'sdisease, Alzheimer's disease, and Parkinson's disease. In someembodiments, the invention provides a method of treating any of theforegoing disorders affecting the nervous system, the method comprisingadministering a complement inhibitor according to a dosing scheduledescribed herein to a subject in need of treatment for the disorder.

In some embodiments, a chronic complement-mediated disorder affects thecirculatory system. For example, in some embodiments the disorder is avasculitis or other disorder associated with vessel inflammation, e.g.,blood vessel and/or lymph vessel inflammation. In some embodiments, avasculitis is polyarteritis nodosa, Wegener's granulomatosis, giant cellarteritis, Churg-Strauss syndrome, microscopic polyangiitis,Henoch-Schonlein purpura, Takayasu's arteritis, Kawasaki disease, orBehcet's disease. In some embodiments, a subject, e.g., a subject inneed of treatment for vasculitis, is positive for antineutrophilcytoplasmic antibody (ANCA).

In some embodiments, a chronic complement-mediated disorder affects thegastrointestinal system. For example, the disorder may be inflammatorybowel disease, e.g., Crohn's disease or ulcerative colitis. In someembodiments, the invention provides a method of treating a chroniccomplement-mediated disorder that affects the gastrointestinal system,the method comprising administering a complement inhibitor according toa dosing schedule described herein to a subject in need of treatment forthe disorder.

In some embodiments, a chronic complement-mediated disorder is athyroiditis (e.g., Hashimoto's thryoiditis, Graves' disease, post-partumthryoiditis), myocarditis, hepatitis (e.g., hepatitis C), pancreatitis,glomerulonephritis (e.g., membranoproliferative glomerulonephritis ormembranous glomerulonephritis), or panniculitis.

In some embodiments, the invention provides methods of treating asubject suffering from chronic pain, the methods comprisingadministering a complement inhibitor to a subject according to a dosingschedule of the present invention. In some embodiments, a subjectsuffers from neuropathic pain. Neuropathic pain has been defined as paininitiated or caused by a primary lesion or dysfunction in the nervoussystem, in particular, pain arising as a direct consequence of a lesionor disease affecting the somatosensory system. For example, neuropathicpain may arise from lesions that involve the somatosensory pathways withdamage to small fibres in peripheral nerves and/or to thespino-thalamocortical system in the CNS. In some embodiments,neuropathic pain arises from autoimmune disease (e.g., multiplesclerosis), metabolic disease (e.g., diabetes), infection (e.g., viraldisease such as shingles or HIV), vascular disease (e.g., stroke),trauma (e.g., injury, surgery), or cancer. For example, neuropathic paincan be pain that persists after healing of an injury or after cessationof a stimulus of peripheral nerve endings or pain that arises due todamage to nerves. Exemplary conditions of or associated with neuropathicpain include painful diabetic neuropathy, post-herpetic neuralgia (e.g.,pain persisting or recurring at the site of acute herpes zoster 3 ormore months after the acute episode), trigeminal neuralgia, cancerrelated neuropathic pain, chemotherapy-associated neuropathic pain,HIV-related neuropathic pain (e.g., from HIV neuropathy),central/post-stroke neuropathic pain, neuropathy associated with backpain, e.g., low back pain (e.g., from radiculopathy such as spinal rootcompression, e.g., lumbar root compression, which compression may arisedue to disc herniation), spinal stenosis, peripheral nerve injury pain,phantom limb pain, polyneuropathy, spinal cord injury related pain,myelopathy, and multiple sclerosis. In certain embodiments of theinvention a complement inhibitor is administered according to aninventive dosing schedule to treat neuropathic pain in a subject withone or more of the afore-mentioned conditions.

In some embodiments, a chronic complement-mediated disorder is a chroniceye disorder. In some embodiments, the chronic eye disorder ischaracterized by macular degeneration, choroidal neovascularization(CNV), retinal neovascularization (RNV), ocular inflammation, or anycombination of the foregoing. Macular degeneration, CNV, RNV, and/orocular inflammation may be a defining and/or diagnostic feature of thedisorder. Exemplary disorders that are characterized by one or more ofthese features include, but are not limited to, macular degenerationrelated conditions, diabetic retinopathy, retinopathy of prematurity,proliferative vitreoretinopathy, uveitis, keratitis, conjunctivitis, andscleritis. Macular degeneration related conditions include, e.g.,age-related macular degeneration (AMD). In some embodiments, a subjectis in need of treatment for wet AMD. In some embodiments, a subject isin need of treatment for dry AMD. In some embodiments, a subject is inneed of treatment for geographic atrophy (GA). In some embodiments, asubject is in need of treatment for ocular inflammation. Ocularinflammation can affect a large number of eye structures such as theconjunctiva (conjunctivitis), cornea (keratitis), episclera, sclera(scleritis), uveal tract, retina, vasculature, and/or optic nerve.Evidence of ocular inflammation can include the presence ofinflammation-associated cells such as white blood cells (e.g.,neutrophils, macrophages) in the eye, the presence of endogenousinflammatory mediator(s), one or more symptoms such as eye pain,redness, light sensitivity, blurred vision and floaters, etc. Uveitis isa general term that refers to inflammation in the uvea of the eye, e.g.,in any of the structures of the uvea, including the iris, ciliary bodyor choroid. Specific types of uveitis include iritis, iridocyclitis,cyclitis, pars planitis and choroiditis. In some embodiments, a subjectis in need of treatment for geographic atrophy (GA). In someembodiments, the chronic eye disorder is an eye disorder characterizedby optic nerve damage (e.g., optic nerve degeneration), such asglaucoma.

In some embodiments, a chronic complement-mediated disorder is chronicrejection of a transplanted organ, tissue, cells or populations of cells(collectively “grafts”). Examples of grafts include, e.g., solid organssuch as kidney, liver, lung, pancreas, heart; tissues such as cartilage,tendons, cornea, skin, heart valves, and blood vessels; pancreaticislets or islet cells. Transplant rejection is one of the major risksassociated with transplants between genetically different individuals ofthe same species (allografts) or between individuals of differentspecies (xenografts) and can lead to graft failure and a need to removethe graft from the recipient. As used herein, “chronic rejection” refersto rejection occurring at least 6 months post-transplant, e.g., between6 months and 1, 2, 3, 4, 5 years, or more post-transplant, often aftermonths to years of good graft function. For purposes hereof, chronicrejection can include chronic graft vasculopathy, a term used to referto fibrosis of the internal blood vessels of the transplanted tissue. Insome embodiments, the invention provides a method of treating a subjectin need of treatment to inhibit chronic rejection of a graft, the methodcomprising administering a complement inhibitor to the subject accordingto a dosing schedule described herein. In some embodiments, theinvention provides a method of treating a subject who has undergone atransplant or is scheduled to undergo a transplant within the subsequent12 weeks. In some embodiments, treatment is initiated no later than 1,2, 3, 6, or 12 months following the transplant.

In some aspects, the invention provides a method of treating a subjectin need of treatment for a chronic complement-mediated disorder, e.g., achronic respiratory disorder, the method comprising administeringmultiple doses of a complement inhibitor to the subject according to adosing schedule in which successive doses are administered on average(i) at least 2 weeks after the plasma concentration of the complementinhibitor decreases to no more than 20% of the maximum plasmaconcentration that was reached after the previous dose; (ii) at least 2weeks after plasma complement activation capacity has returned to atleast 50% of baseline or to within the normal range after the previousdose; (iii) at intervals equal to at least 2 times the terminal plasmahalf-life of the complement inhibitor; or (iv) at intervals at least 3weeks apart. In some embodiments, an inventive method comprisesadministration of a complement inhibitor with an average dosing intervalof at least 3 weeks, e.g., between 3 and 15 weeks, e.g., between 3 and12 weeks, e.g., between 3 and 10 weeks, e.g, between 4 and 8 weeks,e.g., about every 4, 5, 6, 7, or 8 weeks. In some embodiments, at least2 of the foregoing conditions are met. In some embodiments, at least 3of the foregoing conditions are met. In some embodiments, all of theforegoing conditions are met.

In certain embodiments of the invention, a complement inhibitor isadministered according to a dosing schedule that is selected based atleast in part on local complement activation capacity and/or localconcentration of the complement inhibitor. For purposes of the presentinvention, “local complement activation capacity” refers to complementactivation capacity in a tissue or organ affected by acomplement-mediated disorder, which may be determined, for example,using a relevant sample obtained from such tissue or organ. For purposesof the present invention, “local concentration”, e.g., localconcentration of a complement inhibitor or a Th17 biomarker such as aTh17-associated cytokine, refers to concentration in a tissue or organ(e.g., a tissue or organ affected by a complement-mediated disorder)which may be determined, for example, using a relevant sample obtainedfrom such tissue or organ. In some embodiments, a sample comprises abody fluid obtained from a tissue or organ (or portion thereof) affectedby a complement-mediated disorder. In some embodiments, a fluid is BALfluid, sputum (e.g., induced sputum), pleural fluid, synovial fluid,vitreous or aqueous humor, or cerebrospinal fluid. The inventionprovides variations of any of the methods described herein, in whichlocal complement activation capacity is used instead of, or in additionto, plasma complement activation capacity. For example, in certainembodiments of the invention, a complement inhibitor is administered atleast 2 weeks after local complement activation capacity has returned toat least 50% of baseline or to within the normal range following theprevious dose. In some embodiments of the invention, a complementinhibitor is administered between 2 and 15 weeks after local complementactivation capacity has returned to at least 50% of baseline or towithin the normal range following the previous dose. In someembodiments, a complement inhibitor is administered according to adosing schedule in which successive doses are administered on average(i) at least 2 weeks after the local concentration of the complementinhibitor decreases to no more than 20% of the maximum localconcentration that was reached after the previous dose In someembodiments of any of the afore-mentioned methods, a complementinhibitor is administered locally.

In some embodiments, an inventive method comprises administration of acomplement inhibitor with an average dosing interval of at least 3weeks, e.g., between 3 and 15 weeks, e.g., between 3 and 12 weeks e.g.,between 3 and 10 weeks, e.g, between 4 and 8 weeks, e.g., about every 4,5, 6, 7, or 8 weeks. In some embodiments, an inventive method comprisesadministration of a complement inhibitor with an average dosing intervalof between 4 and 6 weeks. In some embodiments, a dose sufficient tosubstantially inhibit plasma complement activation capacity isadministered. In some embodiments, a dose sufficient to substantiallyinhibit local complement activation capacity in a tissue or organaffected by a complement-mediated disorder is administered. In someembodiments, complement activation capacity, e.g., plasma complementactivation capacity or local complement activation capacity, isconsidered “substantially inhibited” if reduced to no more than twicebackground levels, e.g., to approximately background levels. Backgroundlevels (e.g., for any aspect or embodiment of the invention) may belevels determined using a variety of suitable approaches. For example, acontrol sample, e.g., a control plasma sample or other body fluidsample, in which complement has been inactivated, e.g., by heatinactivation, or that has been depleted of one or more complementcomponents such as C3 can be used, and/or a control assay can beperformed in which an essential assay component is omitted. In someembodiments, a dose sufficient to reduce and/or maintain plasmacomplement to within the normal range administered. In some embodiments,a dose sufficient to reduce and/or maintain local complement activationin a tissue or organ affected by a complement-mediated disorder towithin the normal range is administered.

In some embodiments of an inventive method, element (i) comprisesadministering multiple doses of a complement inhibitor to the subjectaccording to a dosing schedule in which successive doses areadministered on average at least 2 weeks after the plasma concentrationof the complement inhibitor decreases to no more than 10%, or in someembodiments no more than 5%, or in some embodiments no more than 1%, ofthe maximum plasma concentration that was reached after the previousdose. In some embodiments of an inventive method, element (i) comprisesadministering multiple doses of a complement inhibitor to the subjectaccording to a dosing schedule in which successive doses areadministered on average at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, or 15 weeks after the plasma concentration of the complementinhibitor decreases to no more than 20% of the maximum plasmaconcentration that was reached after the previous dose or, in someembodiments at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15weeks after the plasma concentration of the complement inhibitordecreases to no more than 10%, or in some embodiments no more than 5%,or in some embodiments no more than 1%, of the maximum plasmaconcentration that was reached after the previous dose.

In some embodiments, an inventive method comprises administering acomplement inhibitor at intervals such that the subject's plasmacomplement activation capacity is at least 50% of baseline or within thenormal range for on average at least 2 weeks between doses. In someembodiments, an inventive method comprises administering a complementinhibitor at intervals such that the subject's plasma complementactivation capacity is at least 50% of baseline for on average at least2 weeks between doses. “Baseline” in this context refers to thesubject's typical complement activation capacity when not affected byadministration of an agent or exposure to a stimulus that significantlyaffects the complement system; and not having experienced anexacerbation of asthma or COPD (or, in some aspects of the invention,another complement-mediated disorder, as applicable) within thepreceding 6 weeks. In some embodiments, an inventive dosing regimencomprises administering a complement inhibitor at intervals such thatthe subject's plasma complement activation capacity is within the normalrange for on average at least 2 weeks between doses. “Normal range” inthis context typically refers to a range of within ±2 standarddeviations from a mean value (e.g., an arithmetic mean value) in apopulation of subjects. One of ordinary skill in the art will appreciatethat the specific values for a “normal range” may at least in partdepend on the particular assay used to assess complement activationcapacity and/or factors such as the specific reagents used. In someembodiments, a normal range may be determined using published data. Insome embodiments, a normal range may be appropriately defined by alaboratory, testing center, ordinary skilled artisan, etc.

In some embodiments, the complement inhibitor is administered with adosing interval such that the subject's complement activation capacityis at least 50% of baseline or within the normal range for on average atleast 3 weeks, e.g., between 3 and 15 weeks, e.g., e.g., between 3 and12 weeks, e.g., between 3 and 10 weeks, e.g, between 4 and 8 weeks,e.g., about 4, 5, 6, 7, or 8 weeks between doses. For purposes of thepresent invention, it will be assumed that plasma and serum complementactivation capacity are not significantly different and can be usedinterchangeably absent evidence to the contrary. If a difference isdetermined to exist, the invention provides embodiments in which plasmacomplement activation capacity is used, embodiments in which the serumcomplement activation capacity is used, and embodiments in which anaverage value is used.

In some embodiments, an inventive method comprises administering acomplement at intervals at least equal on average to twice (2×) theplasma half-life of the complement inhibitor when administeredintravenously. In some embodiments, an inventive dosing regimencomprises administering a complement inhibitor at intervals at leastequal on average to 3×, 4×, 5×, 6×, 7×, 8×, 9×, or 10× the plasmahalf-life of the complement inhibitor when administered intravenously.In some embodiments an inventive method comprises administering acomplement inhibitor by a selected administration route, at intervals atleast equal on average to twice (2×) the plasma half-life of thecomplement inhibitor when administered by the same route. In someembodiments, an inventive method comprises administering a complementinhibitor at intervals at least equal on average to 3×, 4×, 5×, 6×, 7×,8×, 9×, or 10× the plasma half-life of the complement inhibitor whenadministered by the same route. In some embodiments, an administrationroute is the respiratory route. In some embodiments, a complementinhibitor has a mean plasma half-life of between 1 and 5 days. In someembodiments, a complement inhibitor has a mean plasma half-life ofbetween 5 and 10 days. In some embodiments, a complement inhibitor has amean plasma half-life of between 10 and 20 days. In some embodiments, acomplement inhibitor has a mean plasma half-life of between 20 and 30days.

It will be appreciated that a variety of approaches to determiningpharmacokinetic (PK) parameters such as half-life can be used. Anappropriate method can be selected by one of ordinary skill in the art.In general, half-life can be determined by a method comprising:administering one or more doses of the compound to subjects, obtainingblood samples from the subject at various times after administration,measuring the concentration of the compound in said samples, andcalculating a half-life based at least in part on said measurements. Forexample, in some embodiments, samples may be obtained at times 0(pre-dose), 5 min, 15 min, 30 min, 1 hr, 4 hr, 8 hr, 24 hr (1 day), 48hr (2 days), 96 hr (4 days), 192 hr (8 days), 14 days, 21 days, 28 dayspost-dose. It will be appreciated that these time points are exemplary.Different time points and/or more or fewer time points could be used invarious embodiments. One of ordinary skill in the art would selectappropriate time points. The blood samples are typically processed toobtain plasma or serum prior to making the measurements. For purposes ofthe present invention, it will be assumed that plasma and serumconcentrations (and pharmacokinetic parameters such as half-life) arenot significantly different and can be used interchangeably absentevidence to the contrary. If a difference is determined to exist, theinvention provides embodiments in which plasma concentrations (and/orplasma half-life) is used, embodiments in which the serum concentrations(and/or serum half-life therein) is used, and embodiments in which anaverage value is used.

One of ordinary skill in the art would select an appropriate method formeasuring the compound. For example, in some embodiments an immunoassayis used. In some embodiments, a chromatography-based method is used(e.g., liquid chromatography (LC), liquid chromatography-massspectrometry (LC-MS) or liquid chromatography-tandem mass spectrometry(LC-MS-MS). In some embodiments, a bioassay is used. In manyembodiments, the half-life is a terminal (elimination) half-life. Insome embodiments, a terminal half-life is calculated followingadministration of a single dose. In some embodiments, a terminalhalf-life is calculated following administration of multiple doses andallowing the concentration to reach steady state. In some embodiments, ahalf-life determined for the initial (distribution) phase is used. Forexample, if the majority of the compound is removed from circulationduring the distribution phase, an initial half-life may be used in someembodiments.

In some embodiments, half-life is determined by conducting a PK analysisusing non-compartmental analysis on multiple dose PK data from a groupof subjects. In some embodiments, half-life is determined by conductinga PK analysis using a standard 1-compartment model on multiple dose PKdata from a group of subjects. In some embodiments, a half-lifedetermined in subjects suffering from a chronic respiratory disorder(e.g., asthma or COPD) is used. In some embodiments, a half-lifedetermined in subjects who are healthy and not known to be sufferingfrom a disorder is used. In some embodiments, a half-life determined insubjects suffering from a complement-mediated disorder other than achronic respiratory disorder is used. In some embodiments, a half-lifedetermined in adults (persons at least 18 years of age) is used.

In some embodiments, half-life is determined using a dose suitable fortreating a chronic complement-mediated disorder, e.g., a chronicrespiratory disorder, e.g., asthma or COPD. In some embodiments, a doseis sufficient to reduce plasma complement activation capacity to no morethan 50% of the lower limit of the normal range. In some embodiments, adose is sufficient to reduce plasma complement activation capacity to nomore than twice background levels, e.g., to approximately backgroundlevels. In some embodiments, half-life is determined using a compositioncomprising the complement inhibitor, wherein the composition is the sameor substantially similar to a composition to be used to treat a chroniccomplement-mediated disorder.

In certain embodiments, a complement inhibitor is modified byconjugation with a polypeptide or non-polypeptide component of use tostabilize the compound, reduce its immunogenicity, increase its lifetimein the body, increase or decrease its solubility, and/or increase itsresistance to degradation. For example, a polymer such as polyethyleneglycol (PEG), albumin, or albumin-binding peptide, may be used. In suchembodiments, “half-life” typically refers to the half-life of thecomplement inhibitor as so modified.

A variety of software tools are available to facilitate calculation ofPK parameters. For example, Phoenix NMLE or Phoenix WinNonlin software(PharSight Corp, St. Louis, Mo.) or Kinetica (Thermo Scientific) can beused. It will be appreciated that a reasonable estimate of half-lifebased on a model can be used. In some embodiments, a half-lifedetermined in a Phase I, II, or III clinical trial of a particularcompound and/or submitted in an application to a regulatory agency suchas the FDA (e.g., an IND or NDA) is used as a half-life in determiningan inventive dosing interval.

In some embodiments, a method comprises administering at least 5, 10,15, 20, or 25 doses are to a subject according to an inventive dosingschedule (i.e., using a dosing interval according to the invention). Insome embodiments, treatment is continued over a period at least 3, 6, 9,12 months, or more, e.g., 1-2 years, 2-5 years, 5-10 years, or more,e.g., indefinitely.

It will be appreciated that minor deviations, such as occasional use ofa shorter or longer dosing interval as compared with a dosing intervalor range specified herein (e.g., up to about 5%, 10%, or 20% of doses,e.g., within a time span such as 6 months, 1 year, etc.) would fallwithin the scope of the invention. In some embodiments, a dosinginterval for a subject may vary over time and/or may be selected atleast in part based on a measurement of complement activation capacityand/or assessment of disease activity (or a biomarkerthereof) betweendoses.

In some embodiments of any of the inventive methods, a complementinhibitor is administered intravenously. In some embodiments of any ofthe inventive methods, a complement inhibitor is administered by therespiratory route. In some embodiments of any of the inventive methods,a complement inhibitor is administered subcutaneously. In someembodiments of any of the inventive methods, a complement inhibitor isadministered intramuscularly. In some embodiments of any of theinventive methods, a complement inhibitor is administered orally.

In some embodiments, a complement inhibitor is administered in aformulation that provides sustained release (also referred to as“extended release” or “controlled release”) of the complement inhibitor.In some embodiments in which a sustained release formulation is used,the time interval between doses is calculated based at least in part onthe length of time that the sustained release formulation releasescomplement inhibitor. For example, if a sustained release formulationreleases a complement inhibitor for N weeks after administration beforebecoming depleted, the invention provides a method of treating a subjectcomprising administering multiple doses of said sustained releaseformulation according to a dosing schedule in which successive doses areadministered with an average dosing interval of at least N+3 weeks,e.g., between N+3 and N+15 weeks, e.g., between N+3 and N+12 weeks,e.g., between N+3 and N+10 weeks, e.g, between N+4 and N+8 weeks, e.g.,about every N+4, N+5, N+6, N+7, or N+8 weeks. In some embodiments, asustained release formulation is considered to be depleted if it nolonger releases sufficient complement inhibitor to maintain thesubject's plasma complement activation capacity and/or local complementactivation capacity (e.g., in a tissue or organ affected by acomplement-mediated disorder) below the normal range or reduced by atleast 50% of baseline. In some embodiments, a sustained releaseformulation is considered to be depleted if it no longer releasessufficient complement inhibitor to maintain the subject's plasmacomplement activation and/or local complement activation (e.g., in atissue or organ affected by a complement-mediated disorder) below thenormal range or reduced by at least 50% of baseline. In someembodiments, a sustained release formulation is considered to bedepleted if at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more ofthe complement inhibitor contained in the formulation when administeredhas been released or the formulation has essentially ceased releasingcomplement inhibitor.

All combinations of the various complement inhibitors, complementinhibitor characteristics (e.g., compound class, molecular weight,half-life, molecular target, etc.), and dosing parameters (e.g., dosinginterval, route of administration, etc.), and disorders, e.g.,respiratory disorders disclosed herein are contemplated in variousembodiments of the invention. For example, in some embodiments, aninventive method comprises intravenous administration of a complementinhibitor with an average dosing interval of at least 3 weeks, e.g.,between 3 and 15 weeks, e.g., between 3 and 12 weeks, e.g., between 3and 10 weeks, e.g, between 4 and 8 weeks, e.g., about every 4, 5, 6, 7,or 8 weeks. In some embodiments, an inventive method comprises pulmonaryadministration of a complement inhibitor with an average dosing intervalof at least 3 weeks, e.g., e.g., between 3 and 15 weeks, e.g., between 3and 12 weeks, e.g., between 3 and 10 weeks, e.g, about every 4, 5, 6, 7,or 8 weeks.

Further provided are methods of selecting a dosing interval foradministering a complement inhibitor. In some embodiments, a method ofselecting a dosing interval for administering a complement inhibitorcomprises (a) obtaining a half-life of the complement inhibitor; and (b)selecting a dosing interval at least 2-10 weeks longer than thehalf-life. In some embodiments, a method of selecting a dosing intervalfor administering a complement inhibitor comprises (a) obtaining ahalf-life of the complement inhibitor; and (b) selecting a dosinginterval at least 3 times as long as the half-life. In some embodiments,a method of selecting a dosing interval for a complement inhibitorcomprises: (a) determining the length of time that the complementinhibitor reduces plasma complement activation capacity by at least 50%of baseline and/or the length of time that the complement inhibitorreducees plasma complement activation capacity to below the normalrange; and (b) selecting any of the inventive dosing intervals set forthabove based on said measured length of time. In some embodiments, amethod of selecting a dosing interval can further comprise testing acomplement inhibitor administered according to an inventive dosingschedule to an animal that serves as a model for a chroniccomplement-mediated disorder, e.g., a chronic complement-mediatedrespiratory disorder.

In some embodiments, an inventive treatment method comprises aninduction phase and a maintenance phase. In many embodiments, theinduction phase (if used) occurs when a subject initiates therapy. Theinduction phase can consist of a period of time during which acomplement inhibitor is administered at a higher dose and/or at morefrequent intervals and/or using a different route of administration thanduring the maintenance phase. During the maintenance phase, thecomplement inhibitor may be administered using any of the inventivedosing schedules and/or dosing intervals described above. For example,the complement inhibitor may be administered weekly during an inductionphase and on average every 4-15 weeks, e.g., every 4-8 weeks, during amaintenance phase. In some embodiments a complement inhibitor isadministered once or more times daily during an induction phase. In someembodiments a complement inhibitor is administered at least 1, 2, 3, 4,5, 6, or 7 times weekly during an induction phase. In some embodimentsan induction phase lasts for up to 1, 2, 3, 4, 5, 6, 7, or 8 weeks. Insome embodiments a dose or dosing interval is adjusted during aninduction phase. For example, in some embodiments the dosing intervalmay be increased over time and/or the dose may be decreased or increasedover time during the induction phase.

As noted above, in some embodiments the chronic respiratory disease isasthma. Information regarding risk factors, epidemiology, pathogenesis,diagnosis, current management of asthma, etc., may be found, e.g., in“Expert Panel Report 3: Guidelines for the Diagnosis and Management ofAsthma”. National Heart Lung and Blood Institute. 2007. (“NHLBIGuidelines”), Global Initiative for Asthma, Global Strategy for AsthmaManagement and Prevention 2010 “GINA Report”) and/or standard textbooksof internal medicine such as Cecil Textbook of Medicine (20th edition),Harrison's Principles of Internal Medicine (17th edition), and/orstandard textbooks focusing on pulmonary medicine. Asthma is a chronicinflammatory disorder of the airways in which many cells and cellularelements play a role, such as, mast cells, eosinophils, T lymphocytes,macrophages, neutrophils, and epithelial cells Asthmatic individualsexperience recurrent episodes associated with symptoms such as wheezing,breathlessness (also termed dyspnea or shortness of breath), chesttightness, and coughing. These episodes are usually associated withwidespread but variable airflow obstruction that is often reversible,either spontaneously or with treatment. The inflammation also causes anassociated increase in the existing bronchial hyperresponsiveness to avariety of stimuli. Airway hyperresponsiveness (an exaggeratedbronchoconstrictor response to stimuli) is a typical feature of asthma.In general, airflow limitation results from bronchoconstriction andairway edema. Reversibility of airflow limitation may be incomplete insome patients with asthma. For example, airway remodeling can lead tofixed airway narrowing. Structural changes can include thickening of thesub-basement membrane, subepithelial fibrosis, airway smooth musclehypertrophy and hyperplasia, blood vessel proliferation and dilation,and mucous gland hyperplasia, and hypersecretion.

Individuals with asthma may experience exacerbations, which areidentified as events characterized by a change from the individual'sprevious status. Severe asthma exacerbations can be defined as eventsthat require urgent action on the part of the individual and his/herphysician to prevent a serious outcome, such as hospitalization or deathfrom asthma. For example, a severe asthma exacerbation may require useof systemic corticosteroids (e.g., oral corticosteroids) in a subjectwhose asthma is usually well controlled without OCS or may require anincrease in a stable maintenance dose. Moderate asthma exacerbations canbe defined as events that are troublesome to the subject, and thatprompt a need for a change in treatment, but that are not severe. Theseevents are clinically identified by being outside the subject's usualrange of day-to-day asthma variation.

Current medications for asthma are typically categorized into twogeneral classes: long-term control medications (“controllermedications”) such as inhaled corticosteroids (ICS), oralcorticosteroids (OCS), long-acting bronchodilators (LABAs), leukotrienemodifiers (e.g., leukotriene receptor antagonists or leukotrienesynthesis inhibitors, anti-IgE antibodies (omalizumab (Xolair®)),cromolyn and nedocromil, which are used to achieve and maintain controlof persistent asthma and quick-relief medications such as short-actingbronchodilators (SABAs), which are used to treat acute symptoms andexacerbations. For purposes of the present invention, these treatmentsmay be referred to as “conventional therapy”. Treatment of exacerbationsmay also include increasing the dose and/or intensity of controllermedication therapy. For example, a course of OCS can be used to regainasthma control. Current guidelines mandate daily administration ofcontroller medication or, in many cases, administration of multipledoses of controller medication each day for subjects with persistentasthma (with the exception of Xolair, which is administered every 2 or 4weeks).

A subject is generally considered to have persistent asthma if thesubject suffers from symptoms on average more than twice a week and/ortypically uses a quick relief medication (e.g., SABA) more than twice aweek for symptom control. “Asthma severity” can be classified based onthe intensity of treatment required to control the subject's asthma oncerelevant comorbidities have been treated and inhaler technique andadherence have been optimized (see, e.g., GINA Report; Taylor, D R, EurRespir J 2008; 32:545-554). The description of treatment intensity canbe based on the medications and doses recommended in the stepwisetreatment algorithm found in guidelines such as NHLBI Guidelines 2007,GINA Report, and their predecessors and/or in standard medicaltextbooks. For example, asthma can be classified as intermittent, mild,moderate, or severe as indicated in Table 1, where “treatment” refers totreatment sufficient to achieve subject's best level of asthma control.(It will be understood that the categories of mild, moderate, and severeasthma in general imply persistent rather than intermittent asthma). Oneof ordinary skill in the art will appreciate that Table 1 is exemplary,and that not all of these medications will be available in allhealthcare systems, which may affect the assessment of asthma severityin some environments. It will also be appreciated that other emerging ornew approaches may affect the classification of mild/moderate asthma.However, the same principle, of mild asthma being defined by the abilityto achieve good control using very low-intensity treatment and severeasthma being defined by the requirement for high-intensity treatment,can still be applied. Asthma severity can also or alternately beclassified based on intrinsic intensity of the disease in the absence oftreatment (see, e.g., NHBLI Guidelines 2007). Assessment can be made onthe basis of current spirometry and the patient's recall of symptomsover the previous 2-4 weeks. Parameters of current impairment and futurerisk may be assessed and included in a determination of the level ofasthma severity. In some embodiments, asthma severity is defined asshown in FIG. 3.4(a), 3.4(b), 3.4(c) of the NHBLI Guidelines, forindividuals 0-4, 5-11, or ≥12 years of age, respectively.

TABLE 1 Treatment-based Asthma Classification Asthma ClassificationTreatment Intermittent SABA as needed (typically no more than twice aweek) Mild Low-dose ICS or other low-intensity treatment (e.g., LTRA,cromolyn, nedocromil, theophylline) Moderate Low to moderate dose ICSand LABA or other extra treatment Severe High-intensity treatment(high-dose ICS and LABA ± oral corticosteroids and/or other extratreatment)

“Asthma control” refers to the extent to which the manifestations ofasthma have been reduced or removed by treatment (whetherpharmacological or non-pharmacological). Asthma control can be assessedbased on factors such as symptom frequency, nighttime symptoms,objective measures of lung function such as spirometry parameters (e.g.,% FEV₁ of predicted, FEV₁ variability, requirement for use of SABA forsymptom control. Parameters of current impairment and future risk may beassessed and included in a determination of the level of asthma control.In some embodiments, asthma control is defined as shown in FIG. 4.3(a),4.3(b), or 4.3(c) of NHBLI Guidelines, for individuals 0-4, 5-11, or ≥12years of age, respectively.

In general, one of ordinary skill in the art can select an appropriatemeans of determining asthma severity level and/or degree of control, andany classification scheme considered reasonable by those of ordinaryskill in the art can be used.

In some embodiments of the invention, a subject suffering frompersistent asthma is treated with a complement inhibitor using aninventive dosing regimen. In some embodiments, the subject suffers frommild or moderate asthma. In some embodiments, the subject suffers fromsevere asthma. In some embodiments, a subject has asthma that is notwell controlled using conventional therapy. In some embodiments, asubject has asthma that, when treated using conventional therapy,requires use of ICS in order to be well controlled. In some embodiments,a subject has asthma that fails to be well controlled despite use ofICS. In some embodiments, a subject has asthma that, if treated usingconventional therapy, would require use of OCS in order to be wellcontrolled. In some embodiments, a subject has asthma that fails to bewell controlled despite use of high intensity conventional therapy thatincludes OCS. In some embodiments of the invention, an inventive dosingregimen comprises administering a complement inhibitor as a controllermedication, wherein the complement inhibitor is administered withreduced frequency and/or on a less regular basis, as compared withstandard controller medications, while maintaining at least equivalentasthma control. In some embodiments, an inventive dosing regimen affordsimproved patient acceptability, compliance, and/or convenience, ascompared with standard regimens of conventional controller medications,while maintaining at least equivalent asthma control. In someembodiments, a subject treated with a complement inhibitor, e.g.,according to an inventive dosing regimen, can significantly decrease thedose (e.g., by at least 50%) or substantially avoid use of ICS, Xolair,and/or OCS as a controller medication.

In some embodiments, the subject suffers from allergic asthma, which isthe case for most asthmatic individuals. In some embodiments, anasthmatic subject is considered to have allergic asthma if anon-allergic trigger for the asthma (e.g., cold, exercise) is not knownand/or is not identified in a standard diagnostic evaluation. In someembodiments, an asthmatic subject is considered to have allergic asthmaif the subject (i) reproducibly develops asthma symptoms (or worseningof asthma symptoms) following exposure to an allergen or allergen(s) towhich the subject is sensitive; (ii) exhibits IgE specific for anallergen or allergen(s) to which the subject is sensitive; (iii)exhibits a positive skin-prick test to an allergen or allergen(s) towhich the subject is sensitive; and/or (iv) exhibits other symptom(s) ofcharacteristic(s) consistent with atopy such as allergic rhinitis,eczema, or elevated total serum IgE. It will be appreciated that aspecific allergic trigger may not be identified but may be suspected orinferred if the subject experiences worsening symptoms in particularenvironments, for example.

Allergen challenge by inhalation is a technique that is widely used inevaluating allergic airway disease. Inhalation of allergen leads tocross-linking of allergen-specific IgE bound to IgE receptors on, e.g.,mast cells and basophils. Activation of secretory pathways ensues,resulting in release of mediators of bronchoconstriction and vascularpermeability. Individuals with allergic asthma may develop variousmanifestations following allergen challenge, e.g., early asthmaticresponse (EAR), late asthmatic response (LAR), airway hyperreactivity(AHR), and airway eosinophilia, each of which can be detected andquantified as known in the art. For example, airway eosiphophilia may bedetected as an increase in eosinophils in sputum and/or BAL fluid. TheEAR, sometimes referred to as the immediate asthmatic response (IAR), isa response to allergen challenge by inhalation that becomes detectableshortly after the inhalation, typically within 10 minutes (min) of theinhalation, e.g., as a decrease in FEV₁. The EAR typically reaches amaximum within 30 min and resolves within 2-3 hours (h) post-challenge.For example, a subject may be considered to exhibit a “positive” EAR ifhis/her FEV₁ decreases by at least 15%, e.g., at least 20%, within thistime window relative to baseline FEV₁ (where “baseline” in this contextrefers to conditions before the challenge, e.g., conditions equivalentto the subject's usual condition when not experiencing an asthmaexacerbation and not exposed to allergic stimuli to which the subject issensitive). The late asthmatic response (LAR) typically starts between 3h and 8 h post-challenge and is characterized by cellular inflammationof the airway, increased bronchiovascular permeability, and mucussecretion. It is typically detected as a decrease in FEV₁, which may begreater in magnitude than that associated with the EAR and potentiallymore clinically important. For example, a subject may be considered toexhibit a “positive” LAR if his/her FEV₁ decreases by at least 15%,e.g., at least 20%, relative to baseline FEV₁ within the relevant timeperiod as compared with baseline FEV₁. A delayed airway response (DAR)may occur beginning between about 26 and 32 h, reaching a maximumbetween about 32 and 48 h and resolving within about 56 h after thechallenge (Pelikan, Z. Ann Allergy Asthma Immunol. 2010,104(5):394-404).

In some embodiments, the chronic respiratory disorder is chronicobstructive pulmonary disease (COPD). COPD encompasses a spectrum ofconditions characterized by airflow limitation that is not fullyreversible even with therapy and is usually progressive. Symptoms ofCOPD include dyspnea (breathlessness), decreased exercise tolerance,cough, sputum production, wheezing, and chest tightness. Persons withCOPD can experience episodes of acute (e.g., developing over course ofless than a week and often over the course of 24 hours or less)worsening of symptoms (termed COPD exacerbations) that can vary infrequency and duration and are associated with significant morbidity.They may be triggered by events such as respiratory infection, exposureto noxious particles, or may have an unknown etiology. Smoking is themost commonly encountered risk factor for COPD, and other inhalationalexposures can also contribute to development and progression of thedisease. The role of genetic factors in COPD is an area of activeresearch. A small percentage of COPD patients have a hereditarydeficiency of alpha-1 antitrypsin, a major circulating inhibitor ofserine proteases, and this deficiency can lead to a rapidly progressiveform of the disease.

Characteristic pathophysiologic features of COPD include narrowing ofand structural changes in the small airways and destruction of lungparenchyma (in particular around alveoli), most commonly due to chronicinflammation. The chronic airflow limitation observed in COPD typicallyinvolves a mixture of these factors, and their relative importance incontributing to airflow limitation and symptoms varies from person toperson. The term “emphysema” refers to enlargement of the air spaces(alveoli) distal to the terminal bronchioles, with destruction of theirwalls. It should be noted that the term “emphysema” is often usedclinically to refer to the medical condition associated with suchpathological changes. Some individuals with COPD have chronicbronchitis, which is defined in clinical terms as a cough with sputumproduction on most days for 3 months of a year, for 2 consecutive years.Further information regarding risk factors, epidemiology, pathogenesis,diagnosis, and current management of COPD may be found, e.g., in “GlobalStrategy for the Diagnosis, Management, and Prevention of ChronicObstructive Pulmonary Disease” (updated 2009) available on the GlobalInitiative on Chronic Obstructive Pulmonary Disease, Inc. (GOLD)website, also referred to herein as the “GOLD Report”, the AmericanThoracic Society/European Respiratory Society Guidelines (2004)available on the ATS website, referred to herein as “ATC/ERS COPDGuidelines” and standard textbooks of internal medicine such as CecilTextbook of Medicine (20.sup.th edition), Harrison's Principles ofInternal Medicine (17th edition), and/or standard textbooks focusing onpulmonary medicine.

In some embodiments methods disclosed herein inhibit (interfere with,disrupt) the DC-Th17-B-Ab-C-DC cycle discussed above. For example,administration of a complement inhibitor may break the cycle by whichcomplement stimulates DC cells to promote the Th17 phenotype. As aresult, the number and/or activity of Th17 cells diminishes, which inturn reduces the amount of Th17-mediated stimulation of B cells andpolyclonal antibody production. In some embodiments, these effectsresult in “resetting” the immunological microenvironment to a morenormal, less pathological state. As described in Example 1, evidencesupporting the capacity of complement inhibition to have a prolongedinhibitory effect on Th17-associated cytokine production was obtained inan animal model of asthma.

In some embodiments, inhibiting the DC-Th17-B-Ab-C-DC cycle has adisease-modifying effect. Without wishing to be bound by any theory,rather than merely treating symptoms of a disorder, inhibiting theDC-Th17-B-Ab-C-DC cycle may interfere with fundamental pathologicmechanisms that may contribute to ongoing tissue damage even whensymptoms are well controlled and/or that may contribute to exacerbationsof the disease. In some embodiments, inhibiting the DC-Th17-B-Ab-C-DCcycle causes a chronic disorder to go into remission. In someembodiments, remission refers to a state of absence or substantialabsence of disease activity in a subject with a chronic disorder, withthe possibility of return of disease. In some embodiments remission maybe sustained for a prolonged period of time (e.g., at least 6 months,e.g., 6-12 months, 12-24 months, or more) in the absence of continuedtherapy or with a reduced dose or increased dosing interval. In someaspects, inhibition of complement may change the immunologicalmicro-environment of a tissue that is rich in Th17 cells and modify itinto a micro-environment that is rich in regulatory T cells (Tregs).Doing so could allow the immune system to “reset” itself and go into astate of remission. In some embodiments, for example, remission may besustained until occurrence of a triggering event. A triggering event maybe, for example, an infection (which may result in production ofpolyclonal antibodies that react both with an infectious agent and aself protein), exposure to particular environmental conditions (e.g.,high levels of air pollutants such as ozone or particulate matter orcomponents of smoke such as cigarette smoke, allergens), etc. Geneticfactors may play a role. For example, individuals having particularalleles of genes encoding complement components may have a higherbaseline level of complement activity, a more reactive complement systemand/or a lower baseline level of endogenous complement regulatoryprotein activity. In some embodiments an individual has a genotypeassociated with increased risk of AMD. For example, the subject may havea polymorphism in a gene encoding a complement protein or complementregulatory protein, e.g., CFH, C3, factor B, wherein the polymorphism isassociated with an increased risk of AMD.

In some embodiments an immunologic microenvironment may becomeprogressively more polarized towards a pathological state over time,e.g., in a subject who has not yet developed symptoms of a chronicdisorder or in a subject who has developed the disorder and has beentreated as described herein. Such a transition may occur stochastically(e.g., due at least in part to apparently random fluctuations inantibody levels and/or affinity) and/or as a result of accumulated“sub-threshold” trigger events that are not of sufficient intensity totrigger a symptomatic outbreak of a disorder.

In some aspects, methods disclosed herein comprise monitoring a subjectfor evidence of the DC-Th17-B-Ab-C-DC cycle. If such evidence isdetected, the subject may be treated with a complement inhibitor and/orother agent that disrupts the DC-Th17-B-Ab-C-DC cycle. In someembodiments a subject is tested for Th17 cells (e.g., Th17 cell numberor relative number) and/or for one or more biomarkers associated withTh17 cells and/or Th17 activity (“Th17 biomarker”). In some embodiments,a subject is treated with a complement inhibitor based at least in parton assessment of Th17 cells with a Th17 biomarker. “Th17 biomarker”encompasses any molecule or detectable indicator that correlates withTh17 cell presence (e.g., number or concentration of Th17 cells) and/orcorrelates with at least one Th17 cell activity. In some embodiments, aTh17 biomarker comprises a level of a Th17-associated cytokine. In someembodiments a Th17-associated cytokine is a cytokine that promotesformation and/or activation of Th17 cells, e.g., IL-6, IL-21, IL-23,and/or IL-1β. In some embodiments a Th17-associated cytokine is acytokine produced by Th17 cells, e.g., IL-17 (e.g., IL-17A and/orIL-17F), IL-21, and/or IL-22. In some embodiments an increased amount orincreased relative amount of a Th17-associated activity is indicative ofincreased Th17 cells and/or increased Th17-associated activity. In someembodiments a relative amount is an amount as compared with a differentcytokine. In some embodiments the different cytokine is associated withTreg cells. In some embodiments the different cytokine is IL-10. In someembodiments levels of 2, 3, 4, 5, or more Th17-associated cytokines aremeasured. A collective index or score indicative of the level ofTh17-associated activity may be obtained and used as a Th17 biomarker.In some embodiments the presence or level of Th17 cells themselves isassessed for any purpose for which a Th17 biomarker may be assessed. Insome embodiments the presence or level of Tregs is assessed. In someembodiments Tregs are identified based on expression of FOXP3.

In some embodiments, a Th17 biomarker level is measured in a sampleobtained from a subject. In some embodiments a sample comprises a bodyfluid, e.g., blood, BAL fluid, sputum, nasal secretion, urine, etc. Insome embodiments a sample comprises a tissue sample, which may beobtained from a tissue or organ affected by a complement-mediateddisorder. In some embodiments two or more samples of different bodyfluids or a body fluid and a tissue sample are assessed. In someembodiments a level is compared with a reference value. In someembodiments a reference value may be a normal value (e.g., a valuewithin a normal range, e.g., an upper limit of a normal range). In someembodiments a reference value may be a value established for the subjectat a previous time, e.g., when the subject's disorder was wellcontrolled or prior to development of the disorder. In some embodiments,if a measured value deviates significantly from a reference value orshows a trend towards increased deviation from a reference value, thesubject may be treated with a complement inhibitor. In some embodimentsthe subject may be treated with a complement inhibitor and a secondagent that disrupts the DC-Th17-B-Ab-C-DC cycle. A “normal range” may bea range that encompasses at least 95% of healthy individuals. In someembodiments a reference value may be a value associated with a disease,e.g., a value typically found in subjects suffering from a disease in anuntreated state. In some embodiments a normal or disease-associatedrange may depend at least in part on demographic factors such as age,sex, etc., and can be adjusted accordingly. An appropriate referencevalue or range may be established empirically for different disordersand/or different Th17 biomarkers and/or, in some embodiments, forindividual subjects.

In some embodiments, in vivo assessment of Th17 cells and/or a Th17biomarker is envisioned. For example, in some embodiments a detectablylabeled agent that binds to Th17 cells (e.g., to a cell surface markeror combination thereof that is reasonably specific for Th17 cells) orthat bind to a Th17-associated cytokine is administered to a subject. Asuitable imaging method is used to visualize the agent in vivo. In someembodiments, for example, an image is obtained of the lungs, skin, orother location that may be affected by a complement-mediated disorder.In some embodiments in vivo detection allows assessment of theimmunological microenvironment in a tissue or organ of interest. In someembodiments a detectable label comprises a fluorescent, radioactive,ultrasound, or magnetically detectable moiety. In some embodiments animaging method comprises magnetic resonance imaging, ultrasound imaging,optical imaging (e.g., fluorescence imaging or bioluminescence imaging),or nuclear imaging. In some embodiments a fluorescent moiety comprises anear-infrared or infrared fluorescent moiety (emitting in thenear-infrared or infrared region of the spectrum). In some embodimentsan imaging method comprises positron emission tomography (PET), andsingle photon emission computed tomography (SPECT) In some embodiments adetectable label is attached to an agent that binds directly to a targetto be detected. In some embodiments a detectable label is associatedwith or incorporated into or comprises particles, which in someembodiments have at their surface an agent that binds directly to atarget to be detected.

In some embodiments, information obtained from a Th17 biomarkerassessment is used together with additional information, e.g., genotypeinformation, environmental exposure information, and/or subjecthistorical information, to determine whether or when to administer acomplement-inhibitor and/or anti-Th17 agent and/or to select a dose ordosing regimen for a subject. In some embodiments any of the biomarkerassessment and/or treatment decision methods may be performed at leastin part by one or more computers. In some embodiments any of thebiomarker assessment and/or treatment decision methods may be embodiedor stored at least in part on a computer-readable medium havingcomputer-executable instructions thereon. In some embodiments acomputer-readable medium comprises any non-transitory and/or tangiblecomputer-readable medium.

In some embodiments retreatment may occur on a fixed time schedule.

Wherever an aspect or embodiment herein is described in relation tocomplement-mediated disorders, analogous aspects and embodimentsrelating to Th17-associated disorders are provided. Wherever an aspector embodiment herein is described in relation to complement-mediateddisorders, analogous aspects and embodiments relating to Th17-associateddisorders are provided. All combinations of the various complementinhibitors, complement inhibitor characteristics (e.g., compound class,molecular weight, half-life, molecular target, etc.), anti-Th17 agents,and dosing parameters (e.g., dosing interval, route of administration,etc.), and disorders disclosed herein are contemplated in variousembodiments. All combinations of the various complement inhibitors,complement inhibitor characteristics (e.g., compound class, molecularweight, half-life, molecular target, etc.), anti-Th17 agents, anti-Th17agent characteristics (e.g., compound class, molecular weight,half-life, molecular target, etc.), and dosing parameters (e.g., dosinginterval, route of administration, etc.), and disorders disclosed hereinare contemplated in various embodiments.

In some aspects, the invention provides methods of treating a chroniccomplement-mediated disorder or Th17-associated disorder comprisingadministering a complement inhibitor and an anti-Th17 agent to a subjectin need thereof. In some embodiments the complement inhibitor and/oranti-Th17 agent are administered according to any suitable dosingregimen. In some embodiments the complement inhibitor and anti-Th17agent are administered according to a dosing regimen described herein.In some embodiments the chronic disorder is any chroniccomplement-mediated disorder or any Th17-associated disorder. In someaspects, the invention provides methods of treating a chroniccomplement-mediated disorder comprising administering an anti-Th17 agentto a subject in need thereof. In some embodiments the anti-Th17 agent isadministered according to any suitable dosing regimen. In someembodiments the anti-Th17 agent is administered according to a dosingregimen described herein. In some embodiments compositions, e.g.,pharmaceutical compositions, comprising a complement inhibitor and ananti-Th17 agent are provided. Exemplary anti-Th17 agents are discussedin Section V.

III. Complement System

In order to facilitate understanding of the invention, and withoutintending to limit the invention in any way, this section provides anoverview of complement and its pathways of activation. Further detailsare found, e.g., in Kuby Immunology, 6^(th) ed., 2006; Paul, W. E.,Fundamental Immunology, Lippincott Williams & Wilkins; 6^(th) ed., 2008;and Walport M J., Complement. First of two parts. N Engl J Med.,344(14):1058-66, 2001.

Complement is an arm of the innate immune system that plays an importantrole in defending the body against infectious agents. The complementsystem comprises more than 30 serum and cellular proteins that areinvolved in three major pathways, known as the classical, alternative,and lectin pathways. The classical pathway is usually triggered bybinding of a complex of antigen and IgM or IgG antibody to C1 (thoughcertain other activators can also initiate the pathway). Activated C1cleaves C4 and C2 to produce C4a and C4b, in addition to C2a and C2b.C4b and C2a combine to form C3 convertase, which cleaves C3 to form C3aand C3b. Binding of C3b to C3 convertase produces C5 convertase, whichcleaves C5 into C5a and C5b. C3a, C4a, and C5a are anaphylotoxins andmediate multiple reactions in the acute inflammatory response. C3a andC5a are also chemotactic factors that attract immune system cells suchas neutrophils.

The alternative pathway is initiated by and amplified at, e.g.,microbial surfaces and various complex polysaccharides. In this pathway,hydrolysis of C3 to C3(H2O), which occurs spontaneously at a low level,leads to binding of factor B, which is cleaved by factor D, generating afluid phase C3 convertase that activates complement by cleaving C3 intoC3a and C3b. C3b binds to targets such as cell surfaces and forms acomplex with factor B, which is later cleaved by factor D, resulting ina C3 convertase. Surface-bound C3 convertases cleave and activateadditional C3 molecules, resulting in rapid C3b deposition in closeproximity to the site of activation and leading to formation ofadditional C3 convertase, which in turn generates additional C3b. Thisprocess results in a cycle of C3 cleavage and C3 convertase formationthat signicantly amplifies the response. Cleavage of C3 and binding ofanother molecule of C3b to the C3 convertase gives rise to a C5convertase. C3 and C5 convertases of this pathway are regulated by hostcell molecules CR1, DAF, MCP, CD59, and fH. The mode of action of theseproteins involves either decay accelerating activity (i.e., ability todissociate convertases), ability to serve as cofactors in thedegradation of C3b or C4b by factor I, or both. Normally the presence ofcomplement regulatory proteins on host cell surfaces preventssignificant complement activation from occurring thereon.

The C5 convertases produced in both pathways cleave C5 to produce C5aand C5b. C5b then binds to C6, C7, and C8 to form C5b-8, which catalyzespolymerization of C9 to form the C5b-9 membrane attack complex (MAC).The MAC inserts itself into target cell membranes and causes cell lysis.Small amounts of MAC on the membrane of cells may have a variety ofconsequences other than cell death.

The lectin complement pathway is initiated by binding of mannose-bindinglectin (MBL) and MBL-associated serine protease (MASP) to carbohydrates.The MB1-1 gene (known as LMAN-1 in humans) encodes a type I integralmembrane protein localized in the intermediate region between theendoplasmic reticulum and the Golgi. The MBL-2 gene encodes the solublemannose-binding protein found in serum. In the human lectin pathway,MASP-1 and MASP-2 are involved in the proteolysis of C4 and C2, leadingto a C3 convertase described above.

Complement activity is regulated by various mammalian proteins referredto as complement control proteins (CCPs) or regulators of complementactivation (RCA) proteins (U.S. Pat. No. 6,897,290). These proteinsdiffer with respect to ligand specificity and mechanism(s) of complementinhibition. They may accelerate the normal decay of convertases and/orfunction as cofactors for factor I, to enzymatically cleave C3b and/orC4b into smaller fragments. CCPs are characterized by the presence ofmultiple (typically 4-56) homologous motifs known as short consensusrepeats (SCR), complement control protein (CCP) modules, or SUSHIdomains, about 50-70 amino acids in length that contain a conservedmotif including four disulfide-bonded cysteines (two disulfide bonds),proline, tryptophan, and many hydrophobic residues. The CCP familyincludes complement receptor type 1 (CR1; C3b:C4b receptor), complementreceptor type 2 (CR2), membrane cofactor protein (MCP; CD46),decay-accelerating factor (DAF), complement factor H (fH), andC4b-binding protein (C4 bp). CD59 is a membrane-bound complementregulatory protein unrelated structurally to the CCPs. Complementregulatory proteins normally serve to limit complement activation thatmight otherwise occur on cells and tissues of the mammalian, e.g., humanhost.

IV. Complement Inhibitors

General

A variety of different complement inhibitors may be used in variousembodiments of the invention. In general, a complement inhibitor canbelong to any of a number of compound classes such as peptides,polypeptides, antibodies, small molecules, and nucleic acids (e.g.,aptamers, RNAi agents such as short interfering RNAs). In certainembodiments a complement inhibitor inhibits an enzymatic activity of acomplement protein. The enzymatic activity may be proteolytic activity,such as ability to cleave another complement protein. In someembodiments, a complement inhibitor inhibits cleavage of C3, C5, orfactor B. In some embodiments, a complement inhibitor acts on C3. Insome embodiments, a complement inhibitor acts on a complement componentthat lies upstream of C3 in the complement activation cascade. In someembodiments, a complement inhibitor inhibits activation or activity ofat least one soluble complement protein produced in the respiratorysystem. In certain embodiments a complement inhibitor that inhibits atleast the classical pathway of complement activation is used. In certainembodiments a complement inhibitor that inhibits both the classical andthe alternative pathway is used. In some embodiments a complementinhibitor that inhibits C3 activation or activity is used. In someembodiments, a complement inhibitor inhibits activation of at least onecomplement receptor protein expressed in the respiratory system. Incertain embodiments the complement receptor protein is a receptor forC3a. In certain embodiments the complement receptor protein is areceptor for C5a.

In some embodiments, a complement inhibitor comprises an antibody thatsubstantially lacks the capacity to activate complement. For example,the antibody may have less than 10%, less than 5%, or less than 1%complement stimulating activity as compared with full length human IgG1.In some embodiments, the antibody comprises a CH2 domain that hasreduced ability to bind C1q as compared with human IgG1 CH2 domain. Insome embodiments, the antibody contains CH1, CH2, and/or CH3 domainsfrom human IgG4 and/or does not contain CH1, CH2, and/or CH3 domainsfrom human IgG1.

In some embodiments, a complement inhibitor used in, e.g., an inventivedosing regimen, has a molecular weight of 1 kD or less. In someembodiments, a complement inhibitor has a molecular weight between 1 kDand 2 kD, between 2 kD and 5 kD, between 5 kD and 10 kD, between 10 kDand 20 kD, between 20 kD and 30 kD, between 30 kD and 50 kD, between 50kD and 100 kD, or between 100 kD and 200 kD.

A complement inhibitor may be at least in part identical to a naturallyoccurring complement inhibiting agent or a variant or fragment thereof.A variety of different omplement inhibiting polypeptides are produced byviruses (e.g., Poxviruses, Herpesviruses), bacteria (e.g.,Staphylococcus), and other microorganisms. Complement inhibitingproteins are produced by various parasites, e.g., ectoparasites, such asticks. A complement inhibitor can comprise at least a portion of amammalian complement control or complement regulatory protein orreceptor. See Ricklin, D., et al. “Complement-targeted Therapeutics”,Nature Biotechnology, 25(11): 1265-75, 2007, for discussion ofcomplement inhibitors that are or have been in preclinical or clinicaldevelopment for various disorders and may be used in various embodimentsof the inventive methods.

In some embodiments a complement inhibitor comprises an adnectin,affibody, anticalin, or other type of polypeptide sometimes used in theart in lieu of an antibody, wherein the polypeptide binds to acomplement component.

The following sections discuss non-limiting exemplary complementinhibitors of use in embodiments of the present invention. Complementinhibitors have been classified in various groups for purposes ofconvenience. It will be understood that certain complement inhibitorsfall into multiple categories.

In some embodiments, a complement inhibitor that binds to substantiallythe same binding site (e.g., a binding site on a complement componentsuch as C3, C5, factor B, factor D, or an active complement splitproduct) as a complement inhibitor described herein is used. In general,the ability of first and second agents to bind to substantially the samesite on a target molecule, such as a complement component or receptor,can be assessed using methods known in the art, such as competitionassays, molecular modeling, etc. (See, e.g., discussion of compstatinanalog mimetics.) Optionally the first and/or second agent can belabeled with a detectable label, e.g., a radiolabel, fluorescent label,etc. Optionally the target molecule, first agent, or second agent isimmobilized on a support, e.g., a slide, filter, chip, beads, etc. Insome embodiments, a second antibody that binds to substantially the samebinding site as a first antibody comprises one or more CDR(s) that areat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to CDR(s) of the first antibody.

Compounds that Inhibit C3 Activation or Activity

Compstatin Analogs and Mimetics

Compstatin is a cyclic peptide that binds to C3 and inhibits complementactivation by, e.g., inhibiting cleavage of C3 to C3a and C3b byconvertase. U.S. Pat. No. 6,319,897 describes a peptide having thesequence Ile-[Cys-Val-Val-Gln-Asp-Trp-Gly-His-His-Arg-Cys]-Thr (SEQ IDNO: 1), with the disulfide bond between the two cysteines denoted bybrackets. It will be understood that the name “compstatin” was not usedin U.S. Pat. No. 6,319,897 but was subsequently adopted in thescientific and patent literature (see, e.g., Morikis, et al., ProteinSci., 7(3):619-27, 1998) to refer to a peptide having the same sequenceas SEQ ID NO: 2 disclosed in U.S. Pat. No. 6,319,897, but amidated atthe C terminus as shown in Table 2 (SEQ ID NO: 8). The term “compstatin”is used herein consistently with such usage (i.e., to refer to SEQ IDNO: 8). Compstatin analogs that have higher complement inhibitingactivity than compstatin have been developed. See, e.g., WO2004/026328(PCT/US2003/029653), Morikis, D., et al., Biochem Soc Trans. 32(Pt1):28-32, 2004, Mallik, B., et al., J. Med. Chem., 274-286, 2005;Katragadda, M., et al. J. Med. Chem., 49: 4616-4622, 2006; WO2007062249(PCT/US2006/045539); WO2007044668 (PCT/US2006/039397), WO/2009/046198(PCT/US2008/078593); WO/2010/127336 (PCT/US2010/033345) and discussionbelow.

Compstatin analogs may be acetylated or amidated, e.g., at theN-terminus and/or C-terminus For example, compstatin analogs may beacetylated at the N-terminus and amidated at the C-terminus Consistentwith usage in the art, “compstatin” as used herein, and the activitiesof compstatin analogs described herein relative to that of compstatin,refer to compstatin amidated at the C-terminus (Mallik, 2005, supra).

Concatamers or multimers of compstatin or a complement inhibiting analogthereof are also of use in the present invention.

As used herein, the term “compstatin analog” includes compstatin and anycomplement inhibiting analog thereof. The term “compstatin analog”encompasses compstatin and other compounds designed or identified basedon compstatin and whose complement inhibiting activity is at least 50%as great as that of compstatin as measured, e.g., using any complementactivation assay accepted in the art or substantially similar orequivalent assays. Certain suitable assays are described in U.S. Pat.No. 6,319,897, WO2004/026328, Morikis, supra, Mallik, supra, Katragadda2006, supra, WO2007062249 (PCT/US2006/045539); WO2007044668(PCT/US2006/039397), WO/2009/046198 (PCT/US2008/078593); and/orWO/2010/127336 (PCT/US2010/033345). The assay may, for example, measurealternative or classical pathway-mediated erythrocyte lysis or be anELISA assay. In some embodiments, an assay described in WO/2010/135717(PCT/US2010/035871) is used.

The activity of a compstatin analog may be expressed in terms of itsIC₅₀ (the concentration of the compound that inhibits complementactivation by 50%), with a lower IC₅₀ indicating a higher activity asrecognized in the art. The activity of a preferred compstatin analog foruse in the present invention is at least as great as that of compstatin.It is noted that certain modifications known to reduce or eliminatecomplement inhibiting activity and may be explicitly excluded from anyembodiment of the invention. The IC₅₀ of compstatin has been measured as12 μM using an alternative pathway-mediated erythrocyte lysis assay(WO2004/026328). It will be appreciated that the precise IC₅₀ valuemeasured for a given compstatin analog will vary with experimentalconditions (e.g., the serum concentration used in the assay).Comparative values, e.g., obtained from experiments in which IC₅₀ isdetermined for multiple different compounds under substantiallyidentical conditions, are of use. In one embodiment, the IC₅₀ of thecompstatin analog is no more than the IC₅₀ of compstatin. In certainembodiments of the invention the activity of the compstatin analog isbetween 2 and 99 times that of compstatin (i.e., the analog has an IC₅₀that is less than the IC₅₀ of compstatin by a factor of between 2 and99). For example, the activity may be between 10 and 50 times as greatas that of compstatin, or between 50 and 99 times as great as that ofcompstatin. In certain embodiments of the invention the activity of thecompstatin analog is between 99 and 264 times that of compstatin. Forexample, the activity may be 100, 110, 120, 130, 140, 150, 160, 170,180, 190, 200, 210, 220, 230, 240, 250, 260, or 264 times as great asthat of compstatin. In certain embodiments the activity is between 250and 300, 300 and 350, 350 and 400, or 400 and 500 times as great as thatof compstatin. The invention further contemplates compstatin analogshaving activities between 500 and 1000 times that of compstatin, ormore, e.g., between 1000 and 2000 times that of compstatin, or more. Incertain embodiments the IC₅₀ of the compstatin analog is between about0.2 μM and about 0.5 μM. In certain embodiments the IC₅₀ of thecompstatin analog is between about 0.1 μM and about 0.2 μM. In certainembodiments the IC₅₀ of the compstatin analog is between about 0.05 μMand about 0.1 μM. In certain embodiments the IC₅₀ of the compstatinanalog is between about 0.001 μM and about 0.05 μM.

The K_(d) of compstatin binding to C3 can be measured using isothermaltitration calorimetry (Katragadda, et al., J. Biol. Chem., 279(53),54987-54995, 2004). Binding affinity of a variety of compstatin analogsfor C3 has been correlated with their activity, with a lower K_(d)indicating a higher binding affinity, as recognized in the art. A linearcorrelation between binding affinity and activity was shown for certainanalogs tested (Katragadda, 2004, supra; Katragadda 2006, supra). Incertain embodiments of the invention the compstatin analog binds to C3with a K_(d) of between 0.1 μM and 1.0 μM, between 0.05 μM and 0.1 μM,between 0.025 μM and 0.05 μM, between 0.015 μM and 0.025 μM, between0.01 μM and 0.015 μM, or between 0.001 μM and 0.01 μM.

Compounds “designed or identified based on compstatin” include, but arenot limited to, compounds that comprise an amino acid chain whosesequence is obtained by (i) modifying the sequence of compstatin (e.g.,replacing one or more amino acids of the sequence of compstatin with adifferent amino acid or amino acid analog, inserting one or more aminoacids or amino acid analogs into the sequence of compstatin, or deletingone or more amino acids from the sequence of compstatin); (ii) selectionfrom a phage display peptide library in which one or more amino acids ofcompstatin is randomized, and optionally further modified according tomethod (i); or (iii) identified by screening for compounds that competewith compstatin or any analog thereof obtained by methods (i) or (ii)for binding to C3 or a fragment thereof. Many useful compstatin analogscomprise a hydrophobic cluster, a β-turn, and a disulfide bridge.

In certain embodiments of the invention the sequence of the compstatinanalog comprises or consists essentially of a sequence that is obtainedby making 1, 2, 3, or 4 substitutions in the sequence of compstatin,i.e., 1, 2, 3, or 4 amino acids in the sequence of compstatin isreplaced by a different standard amino acid or by a non-standard aminoacid. In certain embodiments of the invention the amino acid at position4 is altered. In certain embodiments of the invention the amino acid atposition 9 is altered. In certain embodiments of the invention the aminoacids at positions 4 and 9 are altered. In certain embodiments of theinvention only the amino acids at positions 4 and 9 are altered. Incertain embodiments of the invention the amino acid at position 4 or 9is altered, or in certain embodiments both amino acids 4 and 9 arealtered, and in addition up to 2 amino acids located at positionsselected from 1, 7, 10, 11, and 13 are altered. In certain embodimentsof the invention the amino acids at positions 4, 7, and 9 are altered.In certain embodiments of the invention amino acids at position 2, 12,or both are altered, provided that the alteration preserves the abilityof the compound to be cyclized. Such alteration(s) at positions 2 and/or12 may be in addition to the alteration(s) at position 1, 4, 7, 9, 10,11, and/or 13. Optionally the sequence of any of the compstatin analogswhose sequence is obtained by replacing one or more amino acids ofcompstatin sequence further includes up to 1, 2, or 3 additional aminoacids at the C-terminus. In one embodiment, the additional amino acid isGly. Optionally the sequence of any of the compstatin analogs whosesequence is obtained by replacing one or more amino acids of compstatinsequence further includes up to 5, or up to 10 additional amino acids atthe C-terminus. It should be understood that compstatin analogs may haveany one or more of the characteristics or features of the variousembodiments described herein, and characteristics or features of anyembodiment may additionally characterize any other embodiment describedherein, unless otherwise stated or evident from the context. In certainembodiments of the invention the sequence of the compstatin analogcomprises or consists essentially of a sequence identical to that ofcompstatin except at positions corresponding to positions 4 and 9 in thesequence of compstatin.

Compstatin and certain compstatin analogs having somewhat greateractivity than compstatin contain only standard amino acids (“standardamino acids” are glycine, leucine, isoleucine, valine, alanine,phenylalanine, tyrosine, tryptophan, aspartic acid, asparagine, glutamicacid, glutamine, cysteine, methionine, arginine, lysine, proline,serine, threonine and histidine). Certain compstatin analogs havingimproved activity incorporate one or more non-standard amino acids.Useful non-standard amino acids include singly and multiply halogenated(e.g., fluorinated) amino acids, D-amino acids, homo-amino acids,N-alkyl amino acids, dehydroamino acids, aromatic amino acids (otherthan phenylalanine, tyrosine and tryptophan), ortho-, meta- orpara-aminobenzoic acid, phospho-amino acids, methoxylated amino acids,and α,α-disubstituted amino acids. In certain embodiments of theinvention, a compstatin analog is designed by replacing one or moreL-amino acids in a compstatin analog described elsewhere herein with thecorresponding D-amino acid. Such compounds and methods of use thereofare an aspect of the invention. Exemplary non-standard amino acids ofuse include 2-naphthylalanine (2-NaI), 1-naphthylalanine (1-NaI),2-indanylglycine carboxylic acid (2Ig1), dihydrotrpytophan (Dht),4-benzoyl-L-phenylalanine (Bpa), 2-α-aminobutyric acid (2-Abu),3-α-aminobutyric acid (3-Abu), 4-α-aminobutyric acid (4-Abu),cyclohexylalanine (Cha), homocyclohexylalanine (hCha),4-fluoro-L-tryptophan (4fW), 5-fluoro-L-tryptophan (5fW),6-fluoro-L-tryptophan (6fW), 4-hydroxy-L-tryptophan (4OH—W),5-hydroxy-L-tryptophan (5OH—W), 6-hydroxy-L-tryptophan (6OH—W),1-methyl-L-tryptophan (1MeW), 4-methyl-L-tryptophan (4MeW),5-methyl-L-tryptophan (5MeW), 7-aza-L-tryptophan (7aW),α-methyl-L-tryptophan (αMeW), β-methyl-L-tryptophan (βMeW),N-methyl-L-tryptophan (NMeW), ornithine (orn), citrulline, norleucine,γ-glutamic acid, etc.

In certain embodiments of the invention the compstatin analog comprisesone or more Trp analogs (e.g., at position 4 and/or 7 relative to thesequence of compstatin). Exemplary Trp analogs are mentioned above. Seealso Beene, et. al. Biochemistry 41: 10262-10269, 2002 (describing,inter alia, singly- and multiply-halogenated Trp analogs); Babitzke &Yanofsky, J. Biol. Chem. 270: 12452-12456, 1995 (describing, inter alia,methylated and halogenated Trp and other Trp and indole analogs); andU.S. Pat. Nos. 6,214,790, 6,169,057, 5,776,970, 4,870,097, 4,576,750 and4,299,838. Other Trp analogs include variants that are substituted(e.g., by a methyl group) at the α or β carbon and, optionally, also atone or more positions of the indole ring Amino acids comprising two ormore aromatic rings, including substituted, unsubstituted, oralternatively substituted variants thereof, are of interest as Trpanalogs. In certain embodiments of the invention the Trp analog, e.g.,at position 4, is 5-methoxy, 5-methyl-, 1-methyl-, or1-formyl-tryptophan. In certain embodiments of the invention a Trpanalog (e.g., at position 4) comprising a 1-alkyl substituent, e.g., alower alkyl (e.g., C₁-C₅) substituent is used. In certain embodiments,N(α) methyl tryptophan or 5-methyltryptophan is used. In someembodiments, an analog comprising a 1-alkanyol substituent, e.g., alower alkanoyl (e.g., C₁-C₅) is used. Examples include1-acetyl-L-tryptophan and L-β-tryptophan.

In certain embodiments the Trp analog has increased hydrophobiccharacter relative to Trp. For example, the indole ring may besubstituted by one or more alkyl (e.g., methyl) groups. In certainembodiments the Trp analog participates in a hydrophobic interactionwith C3. Such a Trp analog may be located, e.g., at position 4 relativeto the sequence of compstatin. In certain embodiments the Trp analogcomprises a substituted or unsubstituted bicyclic aromatic ringcomponent or two or more substituted or unsubstituted monocyclicaromatic ring components.

In certain embodiments the Trp analog has increased propensity to formhydrogen bonds with C3 relative to Trp but does not have increasedhydrophobic character relative to Trp. The Trp analog may have increasedpolarity relative to Trp and/or an increased ability to participate inan electrostatic interaction with a hydrogen bond donor on C3. Certainexemplary Trp analogs with an increased hydrogen bond forming charactercomprise an electronegative substituent on the indole ring. Such a Trpanalog may be located, e.g., at position 7 relative to the sequence ofcompstatin.

In certain embodiments of the invention the compstatin analog comprisesone or more Ala analogs (e.g., at position 9 relative to the sequence ofcompstatin), e.g., Ala analogs that are identical to Ala except thatthey include one or more CH₂ groups in the side chain. In certainembodiments the Ala analog is an unbranched single methyl amino acidsuch as 2-Abu. In certain embodiments of the invention the compstatinanalog comprises one or more Trp analogs (e.g., at position 4 and/or 7relative to the sequence of compstatin) and an Ala analog (e.g., atposition 9 relative to the sequence of compstatin).

In certain embodiments of the invention the compstatin analog is acompound that comprises a peptide that has a sequence of(X′aa)_(n)-Gln-Asp-Xaa-Gly-(X″aa)_(m), (SEQ ID NO: 2) wherein each X′aaand each X″aa is an independently selected amino acid or amino acidanalog, wherein Xaa is Trp or an analog of Trp, and wherein n>1 and m>1and n+m is between 5 and 21. The peptide has a core sequence ofGln-Asp-Xaa-Gly (SEQ ID NO: 71), where Xaa is Trp or an analog of Trp,e.g., an analog of Trp having increased propensity to form hydrogenbonds with an H-bond donor relative to Trp but, in certain embodiments,not having increased hydrophobic character relative to Trp. For example,the analog may be one in which the indole ring of Trp is substitutedwith an electronegative moiety, e.g., a halogen such as fluorine. In oneembodiment Xaa is 5-fluorotryptophan. Absent evidence to the contrary,one of skill in the art would recognize that any non-naturally occurringpeptide whose sequence comprises this core sequence and that inhibitscomplement activation and/or binds to C3 will have been designed basedon the sequence of compstatin. In an alternative embodiment Xaa is anamino acid or amino acid analog other than a Trp analog that allows theGln-Asp-Xaa-Gly (SEQ ID NO: 71) peptide to form a β-turn.

In certain embodiments of the invention the peptide has a core sequenceof X′aa-Gln-Asp-Xaa-Gly (SEQ ID NO: 3), where X′aa and Xaa are selectedfrom Trp and analogs of Trp. In certain embodiments of the invention thepeptide has a core sequence of X′aa-Gln-Asp-Xaa-Gly (SEQ ID NO: 3),where X′aa and Xaa are selected from Trp, analogs of Trp, and otheramino acids or amino acid analogs comprising at least one aromatic ring.In certain embodiments of the invention the core sequence forms a β-turnin the context of the peptide. The β-turn may be flexible, allowing thepeptide to assume two or more conformations as assessed for example,using nuclear magnetic resonance (NMR). In certain embodiments X′aa isan analog of Trp that comprises a substituted or unsubstituted bicyclicaromatic ring component or two or more substituted or unsubstitutedmonocyclic aromatic ring components. In certain embodiments of theinvention X′aa is selected from the group consisting of2-napthylalanine, 1-napthylalanine, 2-indanylglycine carboxylic acid,dihydrotryptophan, and benzoylphenylalanine. In certain embodiments ofthe invention X′aa is an analog of Trp that has increased hydrophobiccharacter relative to Trp. For example, X′aa may be 1-methyltryptophan.In certain embodiments of the invention Xaa is an analog of Trp that hasincreased propensity to form hydrogen bonds relative to Trp but, incertain embodiments, not having increased hydrophobic character relativeto Trp. In certain embodiments of the invention the analog of Trp thathas increased propensity to form hydrogen bonds relative to Trpcomprises a modification on the indole ring of Trp, e.g., at position 5,such as a substitution of a halogen atom for an H atom at position 5.For example, Xaa may be 5-fluorotryptophan.

In certain embodiments of the invention the peptide has a core sequenceof X′aa-Gln-Asp-Xaa-Gly-X″aa (SEQ ID NO: 4), where X′aa and Xaa are eachindependently selected from Trp and analogs of Trp and X″aa is selectedfrom His, Ala, analogs of Ala, Phe, and Trp. In certain embodiments ofthe invention X′aa is an analog of Trp that has increased hydrophobiccharacter relative to Trp, such as 1-methyltryptophan or another Trpanalog having an alkyl substituent on the indole ring (e.g., at position1, 4, 5, or 6). In certain embodiments X′aa is an analog of Trp thatcomprises a substituted or unsubstituted bicyclic aromatic ringcomponent or two or more substituted or unsubstituted monocyclicaromatic ring components. In certain embodiments of the invention X′aais selected from the group consisting of 2-napthylalanine,1-napthylalanine, 2-indanylglycine carboxylic acid, dihydrotryptophan,and benzoylphenylalanine. In certain embodiments of the invention Xaa isan analog of Trp that has increased propensity to form hydrogen bondswith C3 relative to Trp but, in certain embodiments, not havingincreased hydrophobic character relative to Trp. In certain embodimentsof the invention the analog of Trp that has increased propensity to formhydrogen bonds relative to Trp comprises a modification on the indolering of Trp, e.g., at position 5, such as a substitution of a halogenatom for an H atom at position 5. For example, Xaa may be5-fluorotryptophan. In certain embodiments X″aa is Ala or an analog ofAla such as Abu or another unbranched single methyl amino acid. Incertain embodiments of the invention the peptide has a core sequence ofX′aa-Gln-Asp-Xaa-Gly-X″aa (SEQ ID NO: 4), where X′aa and Xaa are eachindependently selected from Trp, analogs of Trp, and amino acids oramino acid analogs comprising at least one aromatic side chain, and X″aais selected from His, Ala, analogs of Ala, Phe, and Trp. In certainembodiments X″aa is selected from analogs of Trp, aromatic amino acids,and aromatic amino acid analogs.

In certain preferred embodiments of the invention the peptide is cyclic.The peptide may be cyclized via a bond between any two amino acids, oneof which is (X′aa). and the other of which is located within (X″aa)_(m).In certain embodiments the cyclic portion of the peptide is between 9and 15 amino acids in length, e.g., 10-12 amino acids in length. Incertain embodiments the cyclic portion of the peptide is 11 amino acidsin length, with a bond (e.g., a disulfide bond) between amino acids atpositions 2 and 12. For example, the peptide may be 13 amino acids long,with a bond between amino acids at positions 2 and 12 resulting in acyclic portion 11 amino acids in length.

In certain embodiments the peptide comprises or consists of the sequenceX′aa1-X′aa2-X′aa3-X′aa4-Gln-Asp-Xaa-Gly-X″aa1-X″aa2-X″aa3-X″aa4-X″aa5(SEQ ID NO: 5). In certain embodiments X′aa4 and Xaa are selected fromTrp and analogs of Trp, and X′aa1, X′aa2, X′aa3, X″aa1, X″aa2, X″aa3,X″aa4, and X″aa5 are independently selected from among amino acids andamino acid analogs. In certain embodiments X′aa4 and Xaa are selectedfrom aromatic amino acids and aromatic amino acid analogs. Any one ormore of X′aa1, X′aa2, X′aa3, X″aa1, X″aa2, X″aa3, X″aa4, and X″aa5 maybe identical to the amino acid at the corresponding position incompstatin. In one embodiment, X″aa1 is Ala or a single methylunbranched amino acid. The peptide may be cyclized via a covalent bondbetween (i) X′aa1, X′aa2, or X′aa3; and (ii) X″aa2, X″aa3, X″aa4 orX″aa5. In one embodiment the peptide is cyclized via a covalent bondbetween X′aa2 and X″aa4. In one embodiment the covalently bound aminoacid are each Cys and the covalent bond is a disulfide (S—S) bond. Inother embodiments the covalent bond is a C—C, C—O, C—S, or C—N bond. Incertain embodiments one of the covalently bound residues is an aminoacid or amino acid analog having a side chain that comprises a primaryor secondary amine, the other covalently bound residue is an amino acidor amino acid analog having a side chain that comprises a carboxylicacid group, and the covalent bond is an amide bond Amino acids or aminoacid analogs having a side chain that comprises a primary or secondaryamine include lysine and diaminocarboxylic acids of general structureNH₂(CH₂)_(n)CH(NH₂)COOH such as 2,3-diaminopropionic acid (dapa),2,4-diaminobutyric acid (daba), and ornithine (orn), wherein n=1 (dapa),2 (daba), and 3 (orn), respectively. Examples of amino acids having aside chain that comprises a carboxylic acid group include dicarboxylicamino acids such as glutamic acid and aspartic acid. Analogs such asbeta-hydroxy-L-glutamic acid may also be used. In some embodiments apeptide is cyclized with a thioether bond, e.g., as described inPCT/US2011/052442 (WO/2012/040259). For example, in some embodiments adisulfide bond in any of the peptides is replaced with a thioether bond.In some embodiments, a cystathionine is formed. In some embodiments thecystathionine is a delta-cystathionine or a gamma-cystathionine. In someembodiments a modification comprises replacement of a Cys-Cys disulfidebond between cysteines at X′aa2 and X″aa4 in SEQ ID NO: 5 (orcorresponding positions in other sequences) with addition of a CH₂, toform a homocysteine at X′aa2 or X″aa4, and introduction of a thioetherbond, to form a cystathionine. In one embodiment, the cystathionine is agamma-cystathionine. In another embodiment, the cystathionine is adelta-cystathionine. Another modification of use in certain embodimentscomprises replacement of the disulfide bond with a thioether bondwithout the addition of a CH₂, thereby forming a lantithionine. In someembodiments a compstatin analog having a thioether in place of adisulfide bond has increased stability, at least under some conditions,as compared with the compstatin analog having the disulfide bond.

In certain embodiments, the compstatin analog is a compound thatcomprises a peptide having a sequence:

Xaa1-Cys-Val-Xaa2-Gln-Asp-Xaa2*-Gly-Xaa3-His-Arg-Cys-Xaa4 (SEQ ID NO:6); wherein:

-   Xaa1 is Ile, Val, Leu, B¹-Ile, B¹-Val, B¹-Leu or a dipeptide    comprising Gly-Ile or B¹-Gly-Ile, and B¹ represents a first blocking    moiety;-   Xaa2 and Xaa2* are independently selected from Trp and analogs of    Trp;-   Xaa3 is His, Ala or an analog of Ala, Phe, Trp, or an analog of Trp;-   Xaa4 is L-Thr, D-Thr, Ile, Val, Gly, a dipeptide selected from    Thr-Ala and Thr-Asn, or a tripeptide comprising Thr-Ala-Asn, wherein    a carboxy terminal —OH of any of the L-Thr, D-Thr, Ile, Val, Gly,    Ala, or Asn optionally is replaced by a second blocking moiety B²;    and the two Cys residues are joined by a disulfide bond. In some    embodiments, Xaa4 is Leu, Nle, His, or Phe or a depeptide selected    from Xaa5-Ala and Xaa5-Asn, or a tripeptide Xaa5-Ala-Asn, wherein    Xaa5 is selected from Leu, Nle, His or Phe, and wherein a carboxy    terminal —OH of any of the L-Thr, D-Thr, Ile, Val, Gly, Leu, Nle,    His, Phe, Ala, or Asn optionally is replaced by a second blocking    moiety B²; and the two Cys residues are joined by a disulfide bond.

In other embodiments Xaa1 is absent or is any amino acid or amino acidanalog, and Xaa2, Xaa2*, Xaa3, and Xaa4 are as defined above. If Xaa1 isabsent, the N-terminal Cys residue may have a blocking moiety B¹attached thereto.

In another embodiment, Xaa4 is any amino acid or amino acid analog andXaa1, Xaa2, Xaa2*, and Xaa3 are as defined above. In another embodimentXaa4 is a dipeptide selected from the group consisting of: Thr-Ala andThr-Asn, wherein the carboxy terminal —OH or the Ala or Asn isoptionally replaced by a second blocking moiety B².

In any of the embodiments of the compstatin analog of SEQ ID NO: 6, Xaa2may be Trp.

In any of the embodiments of the compstatin analog of SEQ ID NO: 6, Xaa2may be an analog of Trp comprising a substituted or unsubstitutedbicyclic aromatic ring component or two or more substituted orunsubstituted monocyclic aromatic ring components. For example, theanalog of Trp may be selected from 2-naphthylalanine (2-NaI),1-naphthylalanine (1-NaI), 2-indanylglycine carboxylic acid (Ig1),dihydrotrpytophan (Dht), and 4-benzoyl-L-phenylalanine.

In any of the embodiments of the compstatin analog of SEQ ID NO: 6, Xaa2may be an analog of Trp having increased hydrophobic character relativeto Trp. For example, the analog of Trp may be selected from1-methyltryptophan, 4-methyltryptophan, 5-methyltryptophan, and6-methyltryptophan. In one embodiment, the analog of Trp is1-methyltryptophan. In one embodiment, Xaa2 is 1-methyltryptophan, Xaa2*is Trp, Xaa3 is Ala, and the other amino acids are identical to those ofcompstatin.

In any of the embodiments of the compstatin analog of SEQ ID NO: 6,Xaa2* may be an analog of Trp such as an analog of Trp having increasedhydrogen bond forming propensity with C3 relative to Trp, which, incertain embodiments, does not have increased hydrophobic characterrelative to Trp. In certain embodiments the analog of Trp comprises anelectronegative substituent on the indole ring. For example, the analogof Trp may be selected from 5-fluorotryptophan and 6-fluorotryptophan.

In certain embodiments of the invention Xaa2 is Trp and Xaa2* is ananalog of Trp having increased hydrogen bond forming propensity with C3relative to Trp which, in certain embodiments, does not have increasedhydrophobic character relative to Trp. In certain embodiments of thecompstatin analog of SEQ ID NO: 6, Xaa2 is analog of Trp havingincreased hydrophobic character relative to Trp such as an analog of Trpselected from 1-methyltryptophan, 4-methyltryptophan,5-methyltryptophan, and 6-methyltryptophan, and Xaa2* is an analog ofTrp having increased hydrogen bond forming propensity with C3 relativeto Trp which, in certain embodiments, does not have increasedhydrophobic character relative to Trp. For example, in one embodimentXaa2 is methyltryptophan and Xaa2* is 5-fluorotryptophan.

In certain of the afore-mentioned embodiments, Xaa3 is Ala. In certainof the afore-mentioned embodiments Xaa3 is a single methyl unbranchedamino acid, e.g., Abu.

The invention further provides compstatin analogs of SEQ ID NO: 6, asdescribed above, wherein Xaa2 and Xaa2* are independently selected fromTrp, analogs of Trp, and other amino acids or amino acid analogs thatcomprise at least one aromatic ring, and Xaa3 is His, Ala or an analogof Ala, Phe, Trp, an analog of Trp, or another aromatic amino acid oraromatic amino acid analog.

In certain embodiments of the invention the blocking moiety present atthe N- or C-terminus of any of the compstatin analogs described hereinis any moiety that stabilizes a peptide against degradation that wouldotherwise occur in mammalian (e.g., human or non-human primate) blood orinterstitial fluid. For example, blocking moiety B¹ could be any moietythat alters the structure of the N-terminus of a peptide so as toinhibit cleavage of a peptide bond between the N-terminal amino acid ofthe peptide and the adjacent amino acid. Blocking moiety B² could be anymoiety that alters the structure of the C-terminus of a peptide so as toinhibit cleavage of a peptide bond between the C-terminal amino acid ofthe peptide and the adjacent amino acid. Any suitable blocking moietiesknown in the art could be used. In certain embodiments of the inventionblocking moiety B¹ comprises an acyl group (i.e., the portion of acarboxylic acid that remains following removal of the —OH group). Theacyl group typically comprises between 1 and 12 carbons, e.g., between 1and 6 carbons. For example, in certain embodiments of the inventionblocking moiety B¹ is selected from the group consisting of: formyl,acetyl, proprionyl, butyryl, isobutyryl, valeryl, isovaleryl, etc. Inone embodiment, the blocking moiety B¹ is an acetyl group, i.e., Xaa1 isAc-Ile, Ac-Val, Ac-Leu, or Ac-Gly-Ile.

In certain embodiments of the invention blocking moiety B² is a primaryor secondary amine (—NH₂ or —NHR¹, wherein R is an organic moiety suchas an alkyl group).

In certain embodiments of the invention blocking moiety B¹ is any moietythat neutralizes or reduces the positive charge that may otherwise bepresent at the N-terminus at physiological pH. In certain embodiments ofthe invention blocking moiety B² is any moiety that neutralizes orreduces the negative charge that may otherwise be present at theC-terminus at physiological pH.

In certain embodiments of the invention, the compstatin analog isacetylated or amidated at the N-terminus and/or C-terminus,respectively. A compstatin analog may be acetylated at the N-terminus,amidated at the C-terminus, and or both acetylated at the N-terminus andamidated at the C-terminus. In certain embodiments of the invention acompstatin analog comprises an alkyl or aryl group at the N-terminusrather than an acetyl group.

In certain embodiments, the compstatin analog is a compound thatcomprises a peptide having a sequence:

Xaa1-Cys-Val-Xaa2-Gln-Asp-Xaa2*-Gly-Xaa3-His-Arg-Cys-Xaa4 (SEQ ID NO:7); wherein:

-   Xaa1 is Ile, Val, Leu, Ac-Ile, Ac-Val, Ac-Leu or a dipeptide    comprising Gly-Ile or Ac-Gly-Ile;-   Xaa2 and Xaa2* are independently selected from Trp and analogs of    Trp;-   Xaa3 is His, Ala or an analog of Ala, Phe, Trp, or an analog of Trp;-   Xaa4 is L-Thr, D-Thr, Ile, Val, Gly, a dipeptide selected from    Thr-Ala and Thr-Asn, or a tripeptide comprising Thr-Ala-Asn, wherein    a carboxy terminal —OH of any of L-Thr, D-Thr, Ile, Val, Gly, Ala,    or Asn optionally is replaced by —NH₂; and the two Cys residues are    joined by a disulfide bond. In some embodiments, Xaa4 is Leu, Nle,    His, or Phe or a depeptide selected from Xaa5-Ala and Xaa5-Asn, or a    tripeptide Xaa5-Ala-Asn, wherein Xaa5 is selected from Leu, Nle, His    or Phe, and wherein a carboxy terminal —OH of any of the L-Thr,    D-Thr, Ile, Val, Gly, Leu, Nle, His, Phe, Ala, or Asn optionally is    replaced by a second blocking moiety B2; and the two Cys residues    are joined by a disulfide bond.

In some embodiments, Xaa1, Xaa2, Xaa2*, Xaa3, and Xaa4 are as describedabove for the various embodiments of SEQ ID NO: 6. For example, incertain embodiments Xaa2* is Trp. In certain embodiments Xaa2 is ananalog of Trp having increased hydrophobic character relative to Trp,e.g., 1-methyltryptophan. In certain embodiments Xaa3 is Ala. In certainembodiments Xaa3 is a single methyl unbranched amino acid.

In certain embodiments of the invention Xaa1 is Ile and Xaa4 is L-Thr.

In certain embodiments of the invention Xaa1 is Ile, Xaa2* is Trp, andXaa4 is L-Thr.

The invention further provides compstatin analogs of SEQ ID NO: 7, asdescribed above, wherein Xaa2 and Xaa2* are independently selected fromTrp, analogs of Trp, other amino acids or aromatic amino acid analogs,and Xaa3 is His, Ala or an analog of Ala, Phe, Trp, an analog of Trp, oranother aromatic amino acid or aromatic amino acid analog.

In certain embodiments of any of the compstatin analogs describedherein, an analog of Phe is used rather than Phe.

Table 2 provides a non-limiting list of compstatin analogs useful in thepresent invention. The analogs are referred to in abbreviated form inthe left column by indicating specific modifications at designatedpositions (1-13) as compared to the parent peptide, compstatin.Consistent with usage in the art, “compstatin” as used herein, and theactivities of compstatin analogs described herein relative to that ofcompstatin, refer to the compstatin peptide amidated at the C-terminusUnless otherwise indicated, peptides in Table 2 are amidated at theC-terminus Bold text is used to indicate certain modifications. Activityrelative to compstatin is based on published data and assays describedtherein (WO2004/026328, WO2007044668, Mallik, 2005; Katragadda, 2006).Where multiple publications reporting an activity were consulted, themore recently published value is used, and it will be recognized thatvalues may be adjusted in the case of differences between assays. Itwill also be appreciated that in certain embodiments of the inventionthe peptides listed in Table 2 are cyclized via a disulfide bond betweenthe two Cys residues when used in the therapeutic compositions andmethods of the invention. Alternate means for cyclizing the peptides arealso within the scope of the invention. As noted above, in variousembodiments of the invention one or more amino acid(s) of a compstatinanalog (e.g., any of the compstatin analogs disclosed herein) can be anN-alkyl amino acid (e.g., an N-methyl amino acid). For example, andwithout limitation, at least one amino acid within the cyclic portion ofthe peptide, at least one amino acid N-terminal to the cyclic portion,and/or at least one amino acid C-terminal to the cyclic portion may bean N-alkyl amino acid, e.g., an N-methyl amino acid. In some embodimentsof the invention, for example, a compstatin analog comprises an N-methylglycine, e.g., at the position corresponding to position 8 of compstatinand/or at the position corresponding to position 13 of compstatin. Insome embodiments, one or more of the compstatin analogs in Table 2contains at least one N-methyl glycine, e.g., at the positioncorresponding to position 8 of compstatin and/or at the positioncorresponding to position 13 of compstatin. In some embodiments, one ormore of the compstatin analogs in contains at least one N-methylisoleucine, e.g., at the position corresponding to position 13 ofcompstatin. For example, a Thr at or near the C-terminal end of apeptide whose sequence is listed in Table 2 may be replaced by N-methylIle. As will be appreciated, in some embodiments the N-methylated aminoacids comprise N-methyl Gly at position 8 and N-methyl Ile at position13. In some embodiments the N-methylated amino acids comprise N-methylGly in a core sequence such as SEQ ID NO: 3 or SEQ ID NO: 4.

TABLE 2 SEQ ID Activity over Peptide Sequence NO: compstatin CompstatinH-ICVVQDWGHHRCT-CONH2 8 * Ac-compstatin Ac-ICVVQDWGHHRCT-CONH2 9   3 ×more Ac-V4Y/H9A Ac-ICV Y QDWG A HRCT-CONH2 10  14 × more Ac-V4W/H9A -OHAc-ICV W QDWG A HRCT-COOH 11  27 × more Ac-V4W/H9A Ac-ICV W QDWG AHRCT-CONH2 12  45 × more Ac-V4W/H9A/T13dT -OH Ac-ICV W QDWG A HRC dT-COOH 13  55 × more Ac-V4(2-Nal)/H9A Ac-ICV (2-Nal) QDWG A HRCT-CONH2 14 99 × more Ac V4(2-Nal)/H9A -OH Ac-ICV (2-Nal) QDWG A HRCT-COOH 15  38 ×more Ac V4(1-Nal)/H9A -OH Ac-ICV (1-Nal) QDWG A HRCT-COOH 16  30 × moreAc-V42Igl/H9A Ac-ICV(2- Igl) QDWG A HRCT-CONH2 17  39 × moreAc-V42Igl/H9A -OH Ac-ICV(2- Igl) QDWG A HRCT-COOH 18  37 × moreAc-V4Dht/H9A -OH Ac-ICV Dht QDWG A HRCT-COOH 19   5 × moreAc-V4(Bpa)/H9A -OH Ac-ICV (Bpa) QDWG A HRCT-COOH 20  49 × moreAc-V4(Bpa)/H9A Ac-ICV (Bpa) QDWG A HRCT-CONH2 21  86 × moreAc-V4(Bta)/H9A -OH Ac-ICV (Bta) QDWG A HRCT-COOH 22  65 × moreAc-V4(Bta)/H9A Ac-ICV (Bta) QDWG A HRCT-CONH2 23  64 × moreAc-V4W/H9(2-Abu) Ac-ICV W QDWG(2- Abu) HRCT-CONH2 24  64 × more+GN4W/H9A + AN -OH H- G ICV W QDWG A HRCTA N -COOH 25  38 × moreAc-V4(5fW)/H9A Ac-ICV (5fW) QDWG A HRCT-CONH ₂ 26  31 × moreAc-V4(5-MeW)/H9A Ac-ICV (5-methyl-W) QDWG A HRCT-CONH ₂ 27  67 × moreAc-V4(1-MeW)/H9A Ac-ICV (1-meth y l-W) QDWG A HRCT-CONH ₂ 28 264 × moreAc-V4W/W7(5fW)/H9A Ac-ICV W QD (5fW) G A HRCT-CONH ₂ 29 121 × moreAc-V4(5fW)/W7(5fW)/H9A Ac-ICV (5fW) QD( 5fW) G A HRCT-CONH ₂ 30 NAAc-V4(5-MeW)/W7(5fW)H9A Ac-ICV (5-methyl-W) QD (5fW) G A HRCT- 31 NACONH ₂ Ac-V4(1MeW)/W7(5fW)/H9A Ac-ICV(1-methyl-W)QD(5fW)G A HRCT- 32264 × more CONH ₂ +G/V4(6fW)/W7(6fW)H9A + N- H-GICV (6fW) QD(6fW)G AHRCT N -COOH 33 126 × more OH Ac-V4(1-formyl-W)/H9A Ac-ICV( 1-formyl-W)QDWG A HRCT-CONH ₂ 34 264 × more Ac-V4(5-methoxy-W)/H9A Ac-ICV(1-methyoxy-W )QDWG A HRCT- 35  76 × more CONH ₂ GN4(5f-W)/W7(5fW)/H9A +N- H-GICV( 5fW )QD( 5fW )G A HRCT N -COOH 36 112 × more OH NA = notavailable

In certain embodiments of the compositions and methods of the inventionthe compstatin analog has a sequence selected from sequences 9-36. Incertain embodiments of the compositions and methods of the invention thecompstatin analog has a sequence selected from SEQ ID NOs: 14, 21, 28,29, 32, 33, 34, and 36. In certain embodiments of the compositionsand/or methods of the invention the compstatin analog has a sequenceselected from SEQ ID NOs: 30 and 31. In one embodiment of thecompositions and methods of the invention the compstatin analog has asequence of SEQ ID NO: 28. In one embodiment of the compositions andmethods of the invention the compstatin analog has a sequence of SEQ IDNO: 32. In one embodiment of the compositions and methods of theinvention the compstatin analog has a sequence of SEQ ID NO: 34. In oneembodiment of the compositions and methods of the invention thecompstatin analog has a sequence of SEQ ID NO: 36.

In some embodiments a blocking moiety B¹ comprises an amino acid, whichmay be represented as Xaa0. In some embodiments blocking moiety B²comprises an amino acid, which may be represented as XaaN. In someembodiments blocking moiety B¹ and/or B² comprises a non-standard aminoacid, such as a D-amino acid, N-alkyl amino acid (e.g., N-methyl aminoacid). In some embodiments a blocking moiety B¹ and/or B² comprises anon-standard amino acid that is an analog of a standard amino acid. Insome embodiments an amino acid nalog comprises a lower alkyl, loweralkoxy, or halogen substituent, as compared with a standard amino acidof which it is an analog. In some embodiments a substituent is on a sidechain. In some embodiments a substituent is on an alpha carbon atom. Insome embodiments, a blocking moiety B¹ comprising an amino acid, e.g., anon-standard amino acid, further comprises a moiety B^(1a). For example,blocking moiety B¹ may be represented as B^(1a)-Xaa0. In someembodiments B^(1a) neutralizes or reduces a positive charge that mayotherwise be present at the N-terminus at physiological pH. In someembodiments B^(1a) comprises or consists of, e.g., an acyl group that,e.g., comprises between 1 and 12 carbons, e.g., between 1 and 6 carbons.In certain embodiments blocking moiety B^(1a) is selected from the groupconsisting of: formyl, acetyl, proprionyl, butyryl, isobutyryl, valeryl,isovaleryl, etc. In some embodiments, a blocking moiety B² comprising anamino acid, e.g., a non-standard amino acid, may further comprise amoiety B^(2a) For example, blocking moiety B² may be represented asXaaN-B^(2a), where N represents the appropriate number for the aminoacid (which will depend on the numbering used in the rest of thepeptide). In some embodiments B^(2a) neutralizes or reduces a negativecharge that may otherwise be present at the C-terminus at physiologicalpH. In some embodiments B^(2a) comprises or consists of a primary orsecondary amine (e.g., NH₂). It will be understood that a blockingactivity of moiety B^(1a)-Xaa0 and/or XaaN-B^(2a) may be provided byeither or both components of the moiety in various embodiments. In someembodiments a blocking moiety or portion thereof, e.g., an amino acidresidue, may contribute to increasing affinity of the compound for C3 orC3b and/or improve the activity of the compound. In some embodiments acontribution to affinity or activity of an amino acid residue may be atleast as important as a contribution to blocking activity. For example,in some embodiments Xaa0 and/or XaaN in B^(1a)-Xaa0 and/or XaaN-B^(2a)may function mainly to increase affinity or activity of the compound,while B¹a and/or B^(2a) may inhibit digestion of and/or neutralize acharge of the peptide. In some embodiments a compstatin analog comprisesthe amino acid sequence of any of SEQ ID NOs: 5-36, wherein SEQ ID NOs:5-36 is further extended at the N- and/or C-terminus. In someembodiments, the sequence may be represented asB^(1a)-Xaa0-SEQUENCE-XaaN-B^(2a), where SEQUENCE represents any of SEQID NOs: 5-36, wherein B^(1a) and B^(2a) may independently be present orabsent. For example, in some embodiments a compstatin analog comprisesB^(1a)-Xaa0-X′aa1-X′aa2-X′aa3-X′aa4-Gln-Asp-Xaa-Gly-X″aa1-X″aa2-X″aa3-X″aa4-X″aa5-XaaN-B^(2a)(SEQ ID NO: 37), where X′aa1-X′aa2-X′aa3-X′aa4, Xaa, X″aa1, X″aa2,X″aa3, X″aa4, and X″aa5 are as set forth above for SEQ ID NO: 5.

In some embodiments a compstatin analog comprisesB^(1a)-Xaa0-Xaa1-Cys-Val-Xaa2-Gln-Asp-Xaa2*-Gly-Xaa3-His-Arg-Cys-Xaa4-XaaN-B^(2a)(SEQ ID NO: 38), where Xaa1, Xaa2, Xaa2*, Xaa3, and Xaa4 are as setforth above for SEQ ID NO: 6 or wherein Xaa1, Xaa2, Xaa2*, Xaa3, andXaa4 are as set forth for SEQ ID NO: 6 or SEQ ID NO: 7.

In some embodiments a compstatin analog comprisesB^(1a)-Xaa0-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Xaa11-Xaa12-Xaa13-XaaN-B^(2a)(SEQ ID NO: 39) wherein Xaa1, Xaa2, Xaa3, Xaa4, Xaa5, Xaa6, Xaa7, Xaa8,Xaa9, Xaa10, Xaa11, Xaa12, and Xaa13 are identical to amino acids atpositions 1-13 of any of SEQ ID NOs: 9-36.

In some embodiments Xaa0 and/or XaaN in any compstatin analog sequencecomprises an amino acid that comprises an aromatic ring having an alkylsubstituent at one or more positions. In some embodiments an alkylsubstituent is a lower alkyl substituent. For example, in someembodiments an alkyl substituent is a methyl or ethyl group. In someembodiments a substituent is located at any position that does notdestroy the aromatic character of the compound. In some embodiments asubstituent is located at any position that does not destroy thearomatic character of a ring to which the substituent is attached. Insome embodiments a substituent is located at position 1, 2, 3, 4, or 5.In some embodiments Xaa0 comprises an O-methyl analog of tyrosine,2-hydroxyphenylalanine or 3-hydroxyphenylalanine. For purposes of thepresent disclosure, a lower case “m” followed by a three letter aminoacid abbreviation may be used to specifically indicate that the aminoacid is an N-methyl amino acid. For example, where the abbreviation“mGly” appears herein, it denotes N-methyl glycine (also sometimesreferred to as sarcosine or Sar). In some embodiments Xaa0 is orcomprises mGly, Tyr, Phe, Arg, Trp, Thr, Tyr(Me), Cha, mPhe, mVal, mIle,mAla, DTyr, DPhe, DArg, DTrp, DThr, DTyr(Me), mPhe, mVal, mIle, DAla, orDCha. For example, in some embodiments a compstatin analog comprises apeptide having a sequenceB¹-Ile-[Cys-Val-Trp(Me)-Gln-Asp-Trp-mGly-Ala-His-Arg-Cys]-mIle-B² (SEQID NO: 40) orB¹-Ile-[Cys-Val-Trp(Me)-Gln-Asp-Trp-mGly-Ala-His-Arg-Cys]-mIle-B² (SEQID NO: 41). The two Cys residues are joined by a disulfide bond in theactive compounds. In some embodiments the peptide is acetylated at theN-terminus and/or amidated at the C-terminus. In some embodiments B¹comprises B^(1a)-Xaa0 and/or B² comprises XaaN-B^(2a), as describedabove. For example, in some embodiments B¹ comprises or consists of Gly,mGly, Tyr, Phe, Arg, Trp, Thr, Tyr(Me), mPhe, mVal, mIle, mAla, DTyr,DPhe, DTrp, DCha, DAla and B² comprises NH₂, e.g., a carboxy terminal—OH of mIle is replaced by NH₂. In some embodiments B¹ comprises orconsists of mGly, Tyr, DTyr, or Tyr(Me) and B² comprises NH₂, e.g., acarboxy terminal —OH of mIle is replaced by NH₂. In some embodiments anIle at position Xaa1 is replaced by Gly. Complement inhibition potencyand/or C3b binding parameters of selected compstatin analogs aredescribed in WO/2010/127336 (PCT/US2010/033345) and/or in Qu, et al.,Immunobiology (2012), doi:10.1016/j.imbio.2012.06.003.

In some embodiments a blocking moiety or portion thereof, e.g., an aminoacid residue, may contribute to increasing affinity of the compound forC3 or C3b and/or improve the activity of the compound. In someembodiments a contribution to affinity or activity of an amino acid oramino acid analog may be more significant than a blocking activity.

In certain embodiments of the compositions and methods of the inventionthe compstatin analog has a sequence as set forth in Table 2, but wherethe Ac-group is replaced by an alternate blocking moiety B¹, asdescribed herein. In some embodiments the —NH₂ group is replaced by analternate blocking moiety B², as described herein.

In one embodiment, the compstatin analog binds to substantially the sameregion of the β chain of human C3 as does compstatin. In one embodimentthe compstatin analog is a compound that binds to a fragment of theC-terminal portion of the β chain of human C3 having a molecular weightof about 40 kDa to which compstatin binds (Soulika, A. M., et al., Mol.Immunol., 35:160, 1998; Soulika, A. M., et al., Mol. Immunol.43(12):2023-9, 2006). In certain embodiments the compstatin analog is acompound that binds to the binding site of compstatin as determined in acompstatin-C3 structure, e.g., a crystal structure or NMR-derived 3Dstructure. In certain embodiments the compstatin analog is a compoundthat could substitute for compstatin in a compstatin-C3 structure andwould form substantially the same intermolecular contacts with C3 ascompstatin. In certain embodiments the compstatin analog is a compoundthat binds to the binding site of a peptide having a sequence set forthin Table 2, e.g., SEQ ID NO: 14, 21, 28, 29, 32, 33, 34, or 36 oranother compstatin analog sequence disclosed herein in a peptide-C3structure, e.g., a crystal structure. In certain embodiments thecompstatin analog is a compound that binds to the binding site of apeptide having SEQ ID NO: 30 or 31 in a peptide-C3 structure, e.g., acrystal structure. In certain embodiments the compstatin analog is acompound that could substitute for the peptide of SEQ ID NO: 9-36, e.g.,a compound that could substitute for the peptide of SEQ ID NO: 14, 21,28, 29, 32, 33, 34, or 36 or another compstatin analog sequencedisclosed herein in a peptide-C3 structure and would form substantiallythe same intermolecular contacts with C3 as the peptide. In certainembodiments the compstatin analog is a compound that could substitutefor the peptide of SEQ ID NO: 30 or 31 in a peptide-C3 structure andwould form substantially the same intermolecular contacts with C3 as thepeptide.

One of ordinary skill in the art will readily be able to determinewhether a compstatin analog binds to a fragment of the C-terminalportion of the β chain of C3 using routine experimental methods. Forexample, one of skill in the art could synthesize a photocrosslinkableversion of the compstatin analog by including a photo-crosslinking aminoacid such as p-benzoyl-L-phenylalanine (Bpa) in the compound, e.g., atthe C-terminus of the sequence (Soulika, A. M., et al, supra).Optionally additional amino acids, e.g., an epitope tag such as a FLAGtag or an HA tag could be included to facilitate detection of thecompound, e.g., by Western blotting. The compstatin analog is incubatedwith the fragment and crosslinking is initiated. Colocalization of thecompstatin analog and the C3 fragment indicates binding. Surface plasmonresonance may also be used to determine whether a compstatin analogbinds to the compstatin binding site on C3 or a fragment thereof. One ofskill in the art would be able to use molecular modeling softwareprograms to predict whether a compound would form substantially the sameintermolecular contacts with C3 as would compstatin or a peptide havingthe sequence of any of the peptides in Table 2, e.g., SEQ ID NO: 14, 21,28, 29, 32, 33, 34, or 36, or in some embodiments SEQ ID NO: 30 or 31 oranother compstatin analog sequence disclosed herein.

Compstatin analogs may be prepared by various synthetic methods ofpeptide synthesis known in the art via condensation of amino acidresidues, e.g., in accordance with conventional peptide synthesismethods, may be prepared by expression in vitro or in living cells fromappropriate nucleic acid sequences encoding them using methods known inthe art. For example, peptides may be synthesized using standardsolid-phase methodologies as described in Malik, supra, Katragadda,supra, WO2004026328, and/or WO2007062249. Potentially reactive moietiessuch as amino and carboxyl groups, reactive functional groups, etc., maybe protected and subsequently deprotected using various protectinggroups and methodologies known in the art. See, e.g., “Protective Groupsin Organic Synthesis”, 3^(rd) ed. Greene, T. W. and Wuts, P. G., Eds.,John Wiley & Sons, New York: 1999. Peptides may be purified usingstandard approaches such as reversed-phase HPLC. Separation ofdiasteriomeric peptides, if desired, may be performed using knownmethods such as reversed-phase HPLC. Preparations may be lyophilized, ifdesired, and subsequently dissolved in a suitable solvent, e.g., water.The pH of the resulting solution may be adjusted, e.g. to physiologicalpH, using a base such as NaOH. Peptide preparations may be characterizedby mass spectrometry if desired, e.g., to confirm mass and/or disulfidebond formation. See, e.g., Mallik, 2005, and Katragadda, 2006.

A compstatin analog can be modified by addition of a molecule such aspolyethylene glycol (PEG) or similar molecules to stabilize thecompound, reduce its immunogenicity, increase its lifetime in the body,increase or decrease its solubility, and/or increase its resistance todegradation. Methods for pegylation are well known in the art (Veronese,F. M. & Harris, Adv. Drug Deliv. Rev. 54, 453-456, 2002; Davis, F. F.,Adv. Drug Deliv. Rev. 54, 457-458, 2002); Hinds, K. D. & Kim, S. W. Adv.Drug Deliv. Rev. 54, 505-530 (2002; Roberts, M. J., Bentley, M. D. &Harris, J. M. Adv. Drug Deliv. Rev. 54, 459-476; 2002); Wang, Y. S. etal. Adv. Drug Deliv. Rev. 54, 547-570, 2002). A wide variety of polymerssuch as PEGs and modified PEGs, including derivatized PEGs to whichpolypeptides can conveniently be attached are described in NektarAdvanced Pegylation 2005-2006 Product Catalog, Nektar Therapeutics, SanCarlos, Calif., which also provides details of appropriate conjugationprocedures. In another embodiment a compstatin analog is fused to the Fcdomain of an immunoglobulin or a portion thereof. In some otherembodiments a compstatin analog is conjugated to an albumin moiety or toan albumin binding peptide. Thus in some embodiments a compstatin analogis modified with one or more polypeptide or non-polypeptide components,e.g., the compstatin analog is pegylated or conjugated to anothermoiety. In some embodiments the component is not the Fc domain of animmunoglobulin or a portion thereof. A compstatin analog can be providedas a multimer or as part of a supramolecular complex, which can includeeither a single molecular species or multiple different species (e.g.,multiple different analogs).

In some embodiments, a compstatin analog of use in methods describedherein is a long-acting compstatin analog, that has a terminal half-lifeof at least 3, 4, 5, 6, or 7 days. In some embodiments a long-actingcompstatin analog is a pegylated compstatin analog. Exemplarylong-acting compstatin analogs are described below and/or inPCT/US12/37648, entitled “CELL-REACTIVE, LONG-ACTING, OR TARGETEDCOMPSTATIN ANALOGS AND USES THEREOF”, filed May 11, 2012. In someembodiments of any method or composition relating to a compstatinanalog, the compstatin analog comprises a compstatin analog whosesequence comprises any of SEQ ID NOs: 3-41, wherein the compstatinanalog is a long-acting compstatin analog.

In some embodiments, a compstatin analog is a multivalent compoundcomprising a plurality of compstatin analog moieties covalently ornoncovalently linked to a polymeric backbone or scaffold. The compstatinanalog moieties can be identical or different. In certain embodiments ofthe invention the multivalent compound comprises multiple instances, orcopies, of a single compstatin analog moiety. In other embodiments ofthe invention the multivalent compound comprises one or more instancesof each of two of more non-identical compstatin analog moieties, e.g.,3, 4, 5, or more different compstatin analog moieties. In certainembodiments of the invention the number of compstatin analog moieties(“n”) is between 2 and 6. In other embodiments of the invention n isbetween 7 and 20. In other embodiments of the invention n is between 20and 100. In other embodiments n is between 100 and 1,000. In otherembodiments of the invention n is between 1,000 and 10,000. In otherembodiments n is between 10,000 and 50,000. In other embodiments n isbetween 50,000 and 100,000. In other embodiments n is between 100,000and 1,000,000.

The compstatin analog moieties may be attached directly to the polymericscaffold or may be attached via a linking moiety that connects thecompstatin analog moiety to the polymeric scaffold. The linking moietymay be attached to a single compstatin analog moiety and to thepolymeric scaffold. Alternately, a linking moiety may have multiplecompstatin analog moieties joined thereto so that the linking moietyattaches multiple compstatin analog moieties to the polymeric scaffold.

In some embodiments, a compstatin analog comprises an amino acid havinga side chain comprising a primary or secondary amine, e.g., a Lysresidue. For example, any of the compstatin analog sequences disclosedherein may be extended or modified by addition of a linker comprisingone or more amino acids, e.g., one or more amino acids comprising aprimary or secondary amine, e.g., in a side chain thereof. For example,a Lys residue, or a sequence comprising a Lys residue, is added at theN-terminus and/or C-terminus of the compstatin analog. In someembodiments, the Lys residue is separated from the cyclic portion of thecompstatin analog by a rigid or flexible spacer. A linker or spacer may,for example, comprise a substituted or unsubstituted, saturated orunsaturated alkyl chain, oligo(ethylene glycol) chain, and/or othermoieties. The length of the chain may be, e.g., between 2 and 20 carbonatoms. In some embodiments the spacer is or comprises a peptide. Thepeptide spacer may be, e.g., between 1 and 20 amino acids in length,e.g., between 4 and 20 amino acids in length. Suitable spacers cancomprise or consist of multiple Gly residues, Ser residues, or both, forexample. Optionally, the amino acid having a side chain comprising aprimary or secondary amine and/or at least one amino acid in a spacer isa D-amino acid. A PEG moiety or similar molecule or polymeric scaffoldmay be linked to the primary or secondary amine, optionally via alinker. In some embodiments, a bifunctional linker is used. Abifunctional linker may comprise two reactive functional groups, whichmay be the same or different in various embodiments. In variousembodiments, one or more linkers, spacers, and/or techniques ofconjugation described in Hermanson, supra, is used.

Any of a variety of polymeric backbones or scaffolds could be used. Forexample, the polymeric backbone or scaffold may be a polyamide,polysaccharide, polyanhydride, polyacrylamide, polymethacrylate,polypeptide, polyethylene oxide, or dendrimer. Suitable methods andpolymeric backbones are described, e.g., in WO98/46270 (PCT/US98/07171)or WO98/47002 (PCT/US98/06963). In one embodiment, the polymericbackbone or scaffold comprises multiple reactive functional groups, suchas carboxylic acids, anhydride, or succinimide groups. The polymericbackbone or scaffold is reacted with the compstatin analogs. In oneembodiment, the compstatin analog comprises any of a number of differentreactive functional groups, such as carboxylic acids, anhydride, orsuccinimide groups, which are reacted with appropriate groups on thepolymeric backbone. Alternately, monomeric units that could be joined toone another to form a polymeric backbone or scaffold are first reactedwith the compstatin analogs and the resulting monomers are polymerized.In another embodiment, short chains are prepolymerized, functionalized,and then a mixture of short chains of different composition areassembled into longer polymers.

In some aspects a moiety such as a polyethylene glycol (PEG) chain orother polymer(s) that, e.g., stabilize the compound, increase itslifetime in the body, increase its solubility, decrease itsimmunogenicity, and/or increase its resistance to degradation may bereferred to herein as a “clearance reducing moiety” (CRM), and acompstatin analog comprising such a moiety may be referred to as along-acting compstatin analog.

In some aspects, a long-acting compstatin analog comprises a compound offormula M-L-A, wherein A is a moiety that comprises a CRM, L is anoptionally present linking portion, and M comprises a compstatin analogmoiety. The compstatin analog moiety can comprise any compstatin analog,e.g., any compstatin analog described above, in various embodiments.Formula M-L-A encompasses embodiments in which L-A is present at theN-terminus of the compstatin analog moiety, embodiments in which L-A ispresent at the C-terminus of the compstatin analog moiety, embodimentsin which L-A is attached to a side chain of an amino acid of thecompstatin analog moiety, and embodiments where the same or differentL-As are present at both ends of M. It will be appreciated that whencertain compstatin analog(s) are present as a compstatin analog moietyin a compound of formula M-L-A, a functional group of the compstatinanalog will have reacted with a functional group of L to form a covalentbond to A or L. For example, a long-acting compstatin analog in whichthe compstatin analog moiety comprises a compstatin analog that containsan amino acid with a side chain containing a primary amine (NH₂) group(which compstatin analog can be represented by formula R¹—(NH₂)), canhave a formula R¹—NH-L-A in which a new covalent bond to L (e.g., N—C)has been formed and a hydrogen lost. Thus the term “compstatin analogmoiety” includes molecular structures in which at least one atom of acompstatin analog participates in a covalent bond with a second moiety,which may, e.g., modification of a side chain. Similar considerationsapply to compstatin analog moieties present in multivalent compounds. Insome embodiments, a blocking moiety at the N-terminus or C-terminus of acompstatin analog is replaced by L-A in the structure of a long-actingcompstatin analog.

In some embodiments, L comprises an unsaturated moiety such as —CH═CH—or —CH₂—CH═CH—; a moiety comprising a non-aromatic cyclic ring system(e.g., a cyclohexyl moiety), an aromatic moiety (e.g., an aromaticcyclic ring system such as a phenyl moiety); an ether moiety (—C—O—C—);an amide moiety (—C(═O)—N—); an ester moiety (—CO—O—); a carbonyl moiety(—C(═O)—); an imine moiety (—C═N—); a thioether moiety (—C—S—C—); anamino acid residue; and/or any moiety that can be formed by the reactionof two compatible reactive functional groups. In certain embodiments,one or more moieties of a linking portion is/are substituted byindependent replacement of one or more of the hydrogen (or other) atomsthereon with one or more moieties including, but not limited toaliphatic; aromatic, aryl; alkyl, aralkyl, alkanoyl, aroyl, alkoxy;thio; F; Cl; Br; I; —NO2; —CN; —CF3; —CH2CF3; —CHCl2; —CH2OH; —CH2CH2OH;—CH2NH2; —CH2SO2CH3; - or -GRG1 wherein G is —O—, —S—, —NRG2-, —C(═O)—,—S(═O)—, —SO2-, —C(═O)O—, —C(═O)NRG2-, —OC(═O)—, —NRG2C(═O)—, —OC(═O)O—,—OC(═O)NRG2-, —NRG2C(═O)O—, —NRG2C(═O)NRG2-, —C(═S)—, —C(═S)S—,—SC(═S)—, —SC(═S)S—, —C(═NRG2)-, —C(═NRG2)O—, —C(═NRG2)NRG3-,—OC(═NRG2)-, —NRG2C(═NRG3)-, —NRG2SO2-, —NRG2SO2NRG3-, or —SO2NRG2-,wherein each occurrence of RG1, RG2 and RG3 independently includes, butis not limited to, hydrogen, halogen, or an optionally substitutedaliphatic, aromatic, or aryl moiety. It will be appreciated that cyclicring systems when present as substituents may optionally be attached viaa linear moiety. Combinations of substituents and variables envisionedby this invention are preferably those that result in the formation ofstable compounds useful in any one or more of the methods describedherein, e.g., useful for the treatment of one or more disorders and/orfor contacting a cell, tissue, or organ, as described herein, and/oruseful as intermediates in the manufacture of one or more suchcompounds.

L can comprise one or more of any of the moieties described in thepreceding paragraph, in various embodiments. In some embodiments, Lcomprises two or more different moieties linked to one another to form astructure typically having a length of between 1 to about 60 atoms,between 1 to about 50 atoms, e.g., between 1 and 40, between 1 and 30,between 1 and 20, between 1 and 10, or between 1 and 6 atoms, wherelength refers to the number of atoms in the main (longest) chain. Insome embodiments, L comprises two or more different moieties linked toone another to form a structure typically having between 1 to about 40,e.g., between 1 and 30, e.g., between 1 and 20, between 1 and 10, orbetween 1 and 6 carbon atoms in the main (longest) chain.

In some embodiments, a long-acting compstatin analog has an averageplasma half-life of at least 1 day, e.g., 1-3 days, 3-7 days, 7-14 days,or 14-28 days, when administered IV at a dose of 10 mg/kg to humans orto non-human primates. In some embodiments, average plasma half-life ofa long-acting compstatin analog following administration IV at a dose of10 mg/kg to humans or to non-human primates is increased by at least afactor of 2, e.g., by a factor of 2-5, 5-10, 10-50, or 50-100-fold ascompared with that of a corresponding compstatin analog having the sameamino acid sequence (and, if applicable, one or more blockingmoiet(ies)) but not comprising the CRM.

In some embodiments, a plasma half-life is a terminal half-life afteradministration of a single IV dose. In some embodiments, a plasmahalf-life is a terminal half-life after steady state has been reachedfollowing administration of multiple IV doses. In some embodiments, along-acting compstatin analog achieves a Cmax in plasma at least 5-foldas great as that of a corresponding compstatin analog not comprising theCRM, e.g., between 5- and 50-fold as great, following administration ofa single IV dose to a primate, or following administration of multipleIV doses. In some embodiments, a long-acting compstatin analog achievesa Cmax in plasma between 10- and 20-fold as great as that of acorresponding compstatin analog not comprising the CRM followingadministration of a single IV dose to a primate, or followingadministration of multiple IV doses. In some embodiments a primate ishuman. In some embodiments a primate is a non-human primate, e.g., amonkey, such as a Cynomolgus monkey or Rhesus monkey. In someembodiments, renal clearance of a long-acting compstatin analog duringthe first 24 hours following administration IV at a dose of 10 mg/kg tohumans or to non-human primates is reduced by at least a factor of 2,e.g., by a factor of 2-5, 5-10, 10-50, or 50-100-fold as compared withrenal clearance of a corresponding compstatin analog. The concentrationof compstatin analog can be measured in blood and/or urine samplesusing, e.g., UV, HPLC, mass spectrometry (MS) or antibody to the CRM, orcombinations of such methods, such as LC/MS or LC/MS/MS. Pharmacokineticparameters such as half-life and clearance can be determined usingmethods known to those of ordinary skill in the art. Pharmacokineticanalysis can be performed, e.g., with WinNonlin software v 5.2(Pharsight Corporation, St. Louis, Mo.).

In some embodiments, a long-acting compstatin analog has a molaractivity of at least about 10%, 20%, 30%, e.g., between 30% and 40%,between 30% and 50%, between 30% and 60%, between 30% and 70%, between30% and 80%, between 30% and 90%, or more, of the activity of acorresponding compstatin analog having the same amino acid sequence(and, if applicable, one or more blocking moiet(ies)) but not comprisinga CRM. In some embodiments wherein a long-acting compstatin analogcomprises multiple compstatin analog moieties, the molar activity of thelong-acting compstatin analog is at least about 10%, 20%, or 30%, e.g.,between 30% and 40%, between 30% and 50%, between 30% and 60%, between30% and 70%, between 30% and 80%, between 30% and 90%, or more, of thesum of the activities of said compstatin analog moieties. In someembodiments, a polyethylene glycol (PEG) comprises a (CH₂CH₂O)_(m)moiety having a molecular weight of at least 500 daltons. In someembodiments, a linker comprises an (CH₂CH₂O)_(n) moiety having anaverage molecular weight of between about 500; 1,000; 1,500; 2,000;5,000; 10,000; 20,000; 30,000; 40,000; 50,000; 60,000; 70,000; 80,000;90,000; and 100,000 daltons. “Average molecular weight” refers to thenumber average molecular weight. In some embodiments, the polydispersityD of a (CH₂CH₂O)n moiety is between 1.0005 and 1.50, e.g., between 1.005and 1.10, 1.15, 1.20, 1.25, 1.30, 1.40, or 1.50, or any value between1.0005 and 1.50.

In some embodiments, a (CH₂CH₂O)n moiety is monodisperse and thepolydispersity of a (CH₂CH₂O)n moiety is 1.0. Such monodisperse(CH₂CH₂O)n moieties are known in the art and are commercially availablefrom Quanta BioDesign (Powell, Ohio), and include, by way of nonlimitingexample, monodisperse moieties where n is 2, 4, 6, 8, 12, 16, 20, or 24.

In some embodiments, a compound comprises multiple (CH₂CH₂O)_(n)moieties wherein the total molecular weight of said (CH₂CH₂O)_(n)moieties is between about 1,000; 5,000; 10,000; 20,000; 30,000; 40,000;50,000; 60,000; 70,000; 80,000; 90,000; and 100,000 daltons. In someembodiments, the compound comprises multiple (CH₂CH₂O)_(n) moietieshaving defined lengths, e.g., n=4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, or 30 or more. In some embodiments, the compound comprises asufficient number of (CH₂CH₂O)_(n) moieties having defined lengths toresult in a total molecular weight of said (CH₂CH₂O)_(n) moieties ofbetween about 1,000; 5,000; 10,000; 20,000; 30,000; 40,000; 50,000;60,000; 70,000; 80,000; 90,000; and 100,000 daltons. In some embodimentsn is between about 30 and about 3000. In some embodiments a compstatinanalog moiety is attached at each end of a linear PEG. A bifunctionalPEG having a reactive functional group at each end of the chain may beused, e.g., as described above. In some embodiments the reactivefunctional groups are identical while in some embodiments differentreactive functional groups are present at each end. In some embodiments,multiple (CH₂CH₂O)_(n) moieties are provided as a branched structure.The branches may be attached to a linear polymer backbone (e.g., as acomb-shaped structure) or may emanate from one or more central coregroups, e.g., as a star structure. In some embodiments, a branchedmolecule has 3 to 10 (CH₂CH₂O)_(n) chains. In some embodiments, abranched molecule has 4 to 8 (CH₂CH₂O)_(n) chains. In some embodiments,a branched molecule has 10, 9, 8, 7, 6, 5, 4, or 3 (CH₂CH₂O)_(n) chains.In some embodiments, a star-shaped molecule has 10-100, 10-50, 10-30, or10-20 (CH₂CH₂O)_(n) chains emanating from a central core group. In someembodiments a long-acting compstatin analog thus may comprise, e.g.,3-10 compstatin analog moieties, e.g., 4-8 compstatin analog moieties,each attached to a (CH₂CH₂O)_(n) chain via a functional group at the endof the chain. In some embodiments a long-acting compstatin analog maycomprise, e.g., 10-100 compstatin analog moieties, each attached to a(CH₂CH₂O)_(n) chain via a functional group at the end of the chain. Insome embodiments, branches (sometimes referred to as “arms”) of abranched or star-shaped PEG contain about the same number of (CH₂CH₂O)moieties. In some embodiments, at least some of the branch lengths maydiffer. It will be understood that in some embodiments one or more(CH₂CH₂O)_(n) chains does not have a comptatin analog moiety attachedthereto. In some embodiments at least about 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, or 100% of the chains has a compstatin analog moietyattached thereto.

In genera and compounds depicted herein, a polyethylene glycol moiety isdrawn with the oxygen atom on the right side of the repeating unit orthe left side of the repeating unit. In cases where only one orientationis drawn, the present invention encompasses both orientations (i.e.,(CH₂CH₂O)_(n) and (OCH₂CH₂)_(n)) of polyethylene glycol moieties for agiven compound or genus, or in cases where a compound or genus containsmultiple polyethylene glycol moieties, all combinations of orientationsare encompasses by the present disclosure.

Formulas of some exemplary monofunctional PEGs comprising a reactivefunctional group are illustrated below. For illustrative purposes,formulas in which the reactive functional group(s) comprise an NHS esterare depicted, but other reactive functional groups could be used, e.g.,as described above. In some embodiments, the (CH₂CH₂O)_(n) are depictedas terminating at the left end with a methoxy group (OCH₃) but it willbe understood that the chains depicted below and elsewhere herein mayterminate with a different OR moiety (e.g., an aliphatic group, an alkylgroup, a lower alkyl group, or any other suitable PEG end group) or anOH group. It will also be appreciated that moieties other than thosedepicted may connect the (CH₂CH₂O)_(n) moieties with the NHS group invarious embodiments.

In some embodiments, a monofunctional PEG is of formula A:

wherein “Reactive functional group” and n are as defined above anddescribed in classes and subclasses herein;

-   R¹ is hydrogen, aliphatic, or any suitable end group; and-   T is a covalent bond or a C₁₋₁₂ straight or branched, hydrocarbon    chain wherein one or more carbon units of T are optionally and    independently replaced by —O—, —S—, —N(R^(x))—, —C(O)—, —C(O)O—,    —OC(O)—, —N(R^(x))C(O)—, —C(O)N(R^(x))—, —S(O)—, —S(O)₂-,    —N(R^(x))SO₂-, or —SO₂N(R^(x))—; and each R^(x) is independently    hydrogen or C₁₋₆ aliphatic.

Exemplary monofunctional PEGs of formula A include:

In Formula I, the moiety comprising the reactive functional group hasthe general structure —CO—(CH₂)_(m)—COO—NHS, where m=2. In someembodiments, a monofunctional PEGs has the structure of Formula I, wherem is between 1 and 10, e.g., between 1 and 5. For example, in someembodiments m is 3, as shown below:

In Formula II, the moiety comprising the reactive functional group hasthe general structure —(CH₂)_(m)—COO—NHS, where m=1. In some embodimentsa monofunctional PEG has the structure of Formula II, where m is between1 and 10 (e.g., wherein m is 5 as shown in Formula III below), orwherein m is 0 (as shown below in Formula IIIa).

In some embodiments a bifunctional linear PEG comprises a moietycomprising a reactive functional group at each of its ends. The reactivefunctional groups may be the same (homobifunctional) or different(heterobifunctional). In some embodiments the structure of abifunctional PEG may be symmetric, wherein the same moiety is used toconnect the reactive functional group to oxygen atoms at each end of the—(CH₂CH₂O)_(n) chain. In some embodiments different moieties are used toconnect the two reactive functional groups to the PEG portion of themolecule. The structures of exemplary bifunctional PEGs are depictedbelow. For illustrative purposes, formulas in which the reactivefunctional group(s) comprise an NHS ester are depicted, but otherreactive functional groups could be used.

In some embodiments, a bifunctional linear PEG is of formula B:

wherein each T and “Reactive functional group” is independently asdefined above and described in classes and subclasses herein, and n isas defined above and described in classes and subclasses herein.

Exemplary bifunctional PEGs of formula B include:

In Formula IV, the moiety comprising the reactive functional group hasthe general structure —(CH₂)_(m)—COO—NHS, where m=1. In someembodiments, a bifunctional PEGs has the structure of Formula IV, wherem is between 1 and 10, e.g., between 1 and 5.

In Formula V, the moiety comprising the reactive functional group hasthe general structure —CO—(CH₂)_(m)—COO—NHS, where m=2. In someembodiments, a bifunctional PEGs has the structure of Formula V, where mis between 1 and 10, e.g., between 1 and 5.

In some embodiments, a branched, comb, or star-shaped PEG comprises amoiety comprising a reactive functional group at the end of each ofmultiple —(CH₂CH₂O)_(n) chains. The reactive functional groups may bethe same or there may be at least two different groups. In someembodiments, a branched, comb, or star-shaped PEG is of the followingformulae:

wherein each R² is independently a “Reactive functional group” or R¹,and each T, n, and “Reactive functional group” is independently asdefined above and described in classes and subclasses herein. Thestructure of exemplary branched PEGs (having 8 arms, or branches)comprising NHS moieties as reactive functional groups is depicted below:

The structure of exemplary branched PEGs (having 4 arms, or branches)comprising NHS moieties as reactive functional groups is depicted below:

The number of branches emanating from the backbone may be varied. Forexample, the number 4 in the above formulae VI and VII may be changed toany other integer between 0 and 10 in various embodiments. In certainembodiments, one or more branches does not contain a reactive functiongroup and the branch terminates with a —CH₂CH₂OH or —CH₂CH₂OR group, asdescribed above.

In some embodiments a branched PEG has the structure of Formula VII,VIII, or IX (or variants thereof having different numbers of branches)with the proviso that x is

In some embodiments a branched PEG has the structure of Formula VII,VIII, or IX (or variants thereof having different numbers of branches)with the proviso that x

is.

Of course the methylene (CH₂) group in the above x moiety may insteadcomprise a longer alkyl chain (CH2)_(m), where m is up to 2, 3, 4, 5, 6,8, 10, 20, or 30, or may comprise one or more other moieties describedherein.

In some embodiments, exemplary branched PEGs having NHS or maleimdereactive groups are depicted below:

In some embodiments, a variant of Formula X or XI are used, wherein 3 oreach of the 4 branches comprise a reactive functional group.

Still other examples of PEGs may be represented as follows:

As noted above, it will be appreciated that, as described herein, invarious embodiments any of a variety of moieties may be incorporatedbetween the peptide component and (CH₂CH₂O)_(n)—R moiety of along-acting compstatin analog, such as an linear alkyl, ester, amide,aromatic ring (e.g., a substituted or unsubstituted phenyl), asubstituted or unsubstituted cycloalkyl structure, or combinationsthereof. In some embodiments such moiet(ies) may render the compoundmore susceptible to hydrolysis, which may release the peptide portion ofthe compound from the CRM. In some embodiments, such release may enhancethe in vivo tissue penetration and/or activity of the compound. In someembodiments hydrolysis is general (e.g., acid-base) hydrolysis. In someembodiments hydrolysis is enzyme-catalyzed, e.g., esterase-catalyzed. Ofcourse both types of hydrolysis may occur. Examples of PEGs comprisingone or more such moieties and an NHS ester as a reactive functionalgroup are as follows:

In some embodiments a branched (multi-arm) PEG or star-shaped PEGcomprises a pentaerythritol core, hexaglycerin core, ortripentaerythritol core. It will be understood that the branches may notall emanate from a single point in certain embodiments.

Monofunctional, bifunctional, branched, and other PEGs comprising one ormore reactive functional groups may be obtained from, e.g., NOF AmericaCorp. White Plains, NY or BOC Sciences 45-16 Ramsey Road Shirley, NY11967, USA, among others.

In some embodiments a compstatin analog of, e.g., any of SEQ ID NOs:3-41 is extended by one or more amino acids at the N-terminus,C-terminus, or both, wherein at least one of the amino acids has a sidechain that comprises a reactive functional group such as a primary orsecondary amine, a sulfhydryl group, a carboxyl group (which may bepresent as a carboxylate group), a guanidino group, a phenol group, anindole ring, a thioether, or an imidazole ring, wherein the reactivefunctional group may be used, e.g., to attach a CRM or moiety comprisinga CRM. In some embodiments, the amino acid(s) is/are L-amino acids. Insome embodiments, any one or more of the amino acid(s) is a D-aminoacid. If multiple amino acids are added, the amino acids can beindependently selected. In some embodiments, the reactive functionalgroup (e.g., a primary or secondary amine) is used as a target foraddition of a moiety comprising a CRM. Amino acids having a side chainthat comprises a primary or secondary amine include lysine (Lys) anddiaminocarboxylic acids of general structure NH₂(CH₂)_(n)CH(NH₂)COOHsuch as 2,3-diaminopropionic acid (dapa), 2,4-diaminobutyric acid(daba), and ornithine (orn), wherein n=1 (dapa), 2 (daba), and 3 (orn),respectively. In some embodiments at least one amino acid is cysteine,aspartic acid, glutamic acid, arginine, tyrosine, tryptophan,methionine, or histidine. Cysteine has a side chain comprising asulfhydryl group. Aspartic acid and glutamic acid have a side chaincomprising a carboxyl group (ionizable to a carboxylate group). Argininehas a side chain comprising a guanidino group. Tyrosine has a side chaincomprising a phenol group (ionizable to a phenolate group). Tryptophanhas a side chain comprising an indole ring include, e.g., tryptophan.Methionine has a side chain comprising a thioether group include, e.g.,methionine. Histidine has a side chain comprising an imidazole ring. Awide variety of non-standard amino acids having side chains thatcomprise one or more such reactive functional group(s) are available,including naturally occurring amino acids and amino acids not found innature. See, e.g., Hughes, B. (ed.), Amino Acids, Peptides and Proteinsin Organic Chemistry, Volumes 1-4, Wiley-VCH (2009-2011); Blaskovich,M., Handbook on Syntheses of Amino Acids General Routes to Amino Acids,Oxford University Press, 2010. Embodiments in which one or morenon-standard amino acid(s) is/are used to provide a target for additionof a moiety comprising a CRM are encompassed. Any one or more of theamino acid(s) may be protected as appropriate during synthesis of thecompound. For example, one or more amino acid(s) may be protected duringreaction(s) involving the target amino acid side chain. In someembodiments, wherein a sulfhydryl-containing amino acid is used as atarget for addition of a moiety comprising a CRM, the sulfhydryl isprotected while the compound is being cyclized by formation of anintramolecular disulfide bond between other amino acids such ascysteines.

In certain discussion herein, an amino acid having a side chaincontaining an amine group is used as an example. Analogous embodimentsare encompassed in which an amino acid having a side chain containing adifferent reactive functional group is used. In some embodiments, anamino acid having a side chain comprising a primary or secondary amineis attached directly to the N-terminus or C-terminus of any of SEQ IDNOs: 3-41 via a peptide bond. In some embodiments, an amino acid havinga side chain comprising a primary or secondary amine is attached to theN- or C-terminus of any of SEQ ID NOs: 3-41 via a linking portion, whichmay contain any one or more of the linking moieties described above. Insome embodiments, at least two amino acids are appended to either orboth termini. The two or more appended amino acids may be joined to eachother by peptide bonds or at least some of the appended amino acids maybe joined to each other by a linking portion, which may contain any oneor more of the linking moieties described herein.

It will be understood that a corresponding compstatin analog notcomprising the CRM may also lack one or more such amino acids which arepresent in the long-acting compstatin analog to which it corresponds.Thus, a corresponding compstatin analog comprising any of SEQ ID NOs:3-41 and lacking a CRM will be understood to “have the same amino acidsequence” as SEQ ID NO: 3-41, respectively. For example, a correspondingcompstatin analog comprising the amino acid sequence of SEQ ID NO: 14,21, 28, 29, 32, 33, 34, or 36 and lacking a CRM will be understood to“have the same amino acid sequence” as SEQ ID NO: 14, 21, 28, 29, 32,33, 34, or 36, respectively.

For descriptive purposes a peptide having the amino acid sequenceIle-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr (SEQ ID NO:42) (corresponding to the compstatin analog of SEQ ID NO: 28, whereinasterisks in SEQ ID NO: 42 represent cysteines joined by a disulfidebond in the active compound, and (1Me)Trp represents1-methyl-tryptophan)), is used as an exemplary compstatin analog moiety;(CH2)_(n) and (O—CH2-CH2)_(n) are used as examples of linking portions;lysine is used as an example of an amino acid comprising a reactivefunctional group (in some compounds), and acetylation and amidation ofthe N- and C-termini, respectively, are used as optionally presentexemplary blocking moieties in some compounds and may be represented initalics, i.e., as Ac and NH₂ respectively. In some embodiments, SEQ IDNO: 42 is extended to comprise a Lys residue at the N- or C-terminus ofthe peptide, e.g., as exemplified below for a C-terminal linkage:

(SEQ ID NO: 43) Ac-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-Lys-NH ₂.

In some embodiments, a Lys residue is attached to the N- or C-terminusof SEQ ID NO: 42 via a peptide linker, e.g., as exemplified below for aC-terminal linkage:

(SEQ ID NO: 44) Ac-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-(Gly)₅-Lys-NH ₂.

In some embodiments, a linker comprising a primary or secondary amine isadded to the N- or C-terminus of a compstatin analog. In someembodiments, the linker comprises an alkyl chain and/or anoligo(ethylene glycol) moiety. For example, NH₂(CH₂CH₂O)nCH₂C(═O)OH(e.g., 8-amino-3,6-dioxaoctanoic acid (AEEAc) or11-amino-3,6,9-trioxaundecanoic acid) or an NHS ester thereof (e.g., anNHS ester of 8-amino-3,6-dioxaoctanoic acid or11-amino-3,6,9-trioxaundecanoic acid), can be used. In some embodiments,the resulting compound is as follows (wherein the portion contributed bythe linker is shown in bold):

(SEQ ID NO: 45) NH ₂(CH ₂)₅ C(═O)-Ile-Cys-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys-Thr-NH ₂. (SEQ ID NO: 46) NH ₂(CH ₂ CH ₂ O)₂ CH ₂C(═O)-Ile-Cys-Val-(1Me)Trp-Gln- Asp-Trp-Gly-Ala-His-Arg-Cys-Thr-NH ₂

In some embodiments, a Lys residue is attached to the N- or C-terminusof SEQ ID NO: 42 via a linker comprising a non-peptide portion. Forexample, the linker can comprise an alkyl chain, oligo(ethylene glycol)chain, and/or cyclic ring system. In some embodiments, 8-AEEAc or an NHSester thereof is used, resulting (in the case of attachment of Lys atthe C-terminus) in the following compound (wherein the portioncontributed by 8-AEEAc is shown in bold):

(SEQ ID NO: 47) Ac-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-NH-CH ₂ CH ₂ OCH ₂ CH ₂ OCH ₂-C(═O)-Lys-NH ₂

It will be appreciated that in SEQ ID NOs: 45 and 46, a —C(═O) moiety isattached to the adjacent Ile residue via a C—N bond, wherein the N ispart of the amino acid and is not shown. Similarly, in SEQ ID NO: 47, a—C(═O) moiety is attached to the adjacent Lys residue via a C—N bond,wherein the N is part of the amino acid and is not shown. It will alsobe appreciated that that in SEQ ID NO: 47 the NH moiety is attached tothe immediately N-terminal amino acid (Thr), via a C—N bond, wherein theC is the carbonyl carbon of the amino acid and is not shown.

The compounds of SEQ ID NOs: 43-47 can be modified at the primary aminegroup to produce a long-acting compstatin analog.

Exemplary long-acting compstatin analogs are set forth below, wherein nis sufficient to provide an average molecular weight of between about500; 1,000; 1,500; 2,000; 5,000; 10,000; 20,000; 30,000; 40,000; 50,000;60,000; 70,000; 80,000; 90,000; and 100,000 daltons.

(SEQ ID NO: 48) (CH₂CH₂O)_(n)C(═O)-Ile-Cys-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys-Thr-NH ₂) (SEQ ID NO: 49)Ac-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-NH-CH₂CH₂OCH₂CH₂OCH₂-C(═O)-Lys- C(═O)—(CH₂CH₂O)n-NH ₂(SEQ ID NO: 50) Ac-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-Lys-C(═O)—(CH₂CH₂O)n-NH ₂. (SEQ ID NO: 51)Ac-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-(Gly)₅-Lys-C(═O)—(CH₂CH₂O)n—NH ₂ (SEQ ID NO: 52)Ac-(CH₂CH₂O)nC(═O)Lys-(Gly)₅-Ile-Cys*-Va1-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-NH ₂) (SEQ ID NO: 53)Ac-(CH₂CH₂O)nC(═O)Lys-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-NH ₂)

In SEQ ID NO: 48, the (CH₂CH₂O)n is coupled via an amide bond to theN-terminal amino acid. In SEQ ID NOs: 49-53, the (CH₂CH₂O)n moiety iscoupled via an amide bond to a Lys side chain; thus it will beunderstood that the NH₂ at the C-terminus in SEQ ID NOs: 49, 50, and 51,represents amidation of the C-terminus of the peptide, and it will beunderstood that in SEQ ID NOs: 52 and 53, the Ac at the N-terminusrepresents acetylation of the N-terminus of the peptide, as describedabove. It will also be appreciated by those of ordinary skill in the artthat a free end of a (CH₂CH₂O)_(n) moiety typically terminates with an(OR) where the underlined O represents the O atom in the terminal(CH₂CH₂O) group. (OR) is often a moiety such as a hydroxyl (OH) ormethoxy (—OCH₃) group though other groups (e.g., other alkoxy groups)could be used. Thus SEQ ID NO: 49, for example, may be represented asAc-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-NH—CH₂CH₂OCH₂CH₂OCH₂—C(═O)-Lys-(C(═O)—(CH₂CH₂O)_(n)—R)—NH2(SEQ ID NO: 54) wherein R is, e.g., either H or CH₃ in the case of alinear PEG. In the case of a bifunctional, branched or star-shaped PEG,R represents the remainder of the molecule. Further, it will beunderstood that the moiety comprising the reactive functional group mayvary, as described herein (e.g., according to any of the formulasdescribed herein). For example, long-acting compstatin analogscomprising the same peptide sequence as SEQ ID NO: 54, in which themoiety comprising the reactive functional group comprises an esterand/or alkyl chain may be represented as follows

(SEQ ID NO: 55) Ac-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-NH-CH₂CH₂OCH₂CH₂OCH₂-C(═O)-Lys-(C(═O)—(CH₂)_(m)-(CH₂CH₂O)_(n)-R)—NH ₂; (SEQ ID NO: 56)Ac-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-NH-CH₂CH₂OCH₂CH₂OCH₂-C(═O)-Lys-(C(═O)—(CH₂)_(m)C(═O)—(CH₂CH₂O)_(n)-R)-NH ₂ (SEQ ID NO: 57)Ac-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-NH-CH₂CH₂OCH₂CH₂OCH₂-C(═O)-Lys-(C(═O)—(CH₂)_(m)C(═O)—(CH₂)j(CH₂CH₂O)_(n)-R)-NH ₂In SEQ ID NOs: 55-57 m may range from 1 up to about 2, 3, 4, 5, 6, 7, 8,10, 15, 20, or 30 in various embodiments, In SEQ ID NOs: 57 j may rangefrom 1 up to about 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, or 30 in variousembodiments. It will also be appreciated that, as described herein, invarious embodiments other moieties may be incorporated between theLys-(C(═O)— and (CH₂CH₂O)_(n)—R, such as an amide, aromatic ring (e.g.,a substituted or unsubstituted phenyl), or a substituted orunsubstituted cycloalkyl structure.

In some embodiments a long-acting compstatin analog comprises a variantof SEQ ID NOs: 48-57 in which-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-SEQ ID NO:72) is replaced by an amino acid sequence comprising the amino acidsequence of any other compstatin analog, e.g., of any of SEQ ID NOs 3-27or 29-41, with the proviso that blocking moiet(ies) present at the N-and/or C-termini of a compstatin analog may be absent, replaced by alinker (which may comprise a blocking moiety), or attached to adifferent N- or C-terminal amino acid present in the correspondingvariant(s).

Any compstatin analog, e.g., any compound comprising any of SEQ ID NOs:3-41 may be attached via its N-terminus or C-terminus directly orindirectly to any moiety comprising a reactive functional group, e.g.,any of Formulas I-XVI or Compound I-III, in various embodiments.

In some embodiments a CRM comprises a polypeptide that occurs in humanserum, or a fragment thereof or a substantially similar variant of thepolypeptide or fragment thereof. In some embodiments the polypeptide,fragment, or variant has a molecular weight of between 5 kD and 150 kD,e.g., at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 kd, or more,e.g., between 100 and 120, or 120 and 150 kD. In some embodiments,producing a long-acting compstatin analog comprises reacting acompstatin analog comprising a reactive functional group with one ormore amino acid side chains of the polypeptide, wherein the side chaincomprises a compatible functional group. In some embodiments, producinga long-acting compstatin analog comprises reacting a compstatin analogcomprising a reactive functional group with the N-terminal amine and/orC-terminal carboxyl group of the polypeptide. In some embodiments,producing a long-acting compstatin analog comprises reacting acompstatin analog comprising an amine-reactive functional group withamino acids having a side chain comprising a primary amine (e.g.,lysine) and/or with the N-terminal amine of the polypeptide. In someembodiments, producing a long-acting compstatin analog comprisesreacting a compstatin analog comprising a carboxyl-reactive functionalgroup with the C-terminal carboxyl group of the polypeptide. In someembodiments a compstatin analog moiety is attached at each terminus ofthe polypeptide and, optionally, to the side chain of one or moreinternal amino acids. In some embodiments, producing a long-actingcompstatin analog comprises reacting a compstatin analog comprising asulfhydryl-reactive functional group with one or more sulfhydryl groupsof the polypeptide.

In some embodiments, at least one reactive functional group isintroduced into the polypeptide. For example, in some embodiments atleast one side chain of the polypeptide is modified to convert a firstreactive functional group to a different reactive functional group priorto reaction with the compstatin analog. In some embodiments a thiol isintroduced. Several methods are available for introducing thiols intobiomolecules, including the reduction of intrinsic disulfides, as wellas the conversion of amine, aldehyde or carboxylic acid groups to thiolgroups. Disulfide crosslinks of cystines in proteins can be reduced tocysteine residues by dithiothreitol (DTT),tris-(2-carboxyethyl)phosphine (TCEP), or tris-(2-cyanoethyl)phosphineAmines can be indirectly thiolated by reaction with succinimidyl3-(2-pyridyldithio)propionate (SPDP) followed by reduction of the3-(2-pyridyldithio)propionyl conjugate with DTT or TCEP Amines can beindirectly thiolated by reaction with succinimidyl acetylthioacetatefollowed by removal of the acetyl group with 50 mM hydroxylamine orhydrazine at near-neutral pH Amines can be directly thiolated byreaction with 2-iminothiolane, which preserve the overall charge of themolecule and introduces a free thiol. Tryptophan residues in thiol-freeproteins can be oxidized to mercaptotryptophan residues, which can thenbe modified by iodoacetamides or maleimides. A polypeptide comprisingone or more thiols may be reacted with a compstatin analog comprising amaleimide group, such asAc-Ile-Cys*-Val-Trp(1-Me)-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-AEEAc-Lys-(C(═O)—(CH₂)₅-Mal)-NH₂(SEQ ID NO: 58) to generate a long-acting compstatin analog.

In some embodiments the polypeptide is recombinantly produced. In someembodiments the polypeptide is at least in part recombinantly produced(e.g., in bacteria or in eukaryotic host cells such as fungal, insect,plant, or vertebrate) and/or at least in part produced using chemicalsynthesis. In some embodiments the polypeptide is purified. In someembodiments the polypeptide is glycosylated. In some embodiments thepolypeptide is non-glycosylated. In some embodiments the polypeptide ishuman serum albumin (HSA). In some embodiments a substantially similarvariant of the polypeptide is sufficiently similar to the polypeptide ofwhich it is a variant so as to not be recognized as foreign by a normalimmune system of a subject, e.g., a human subject. In some embodimentsalterations in the sequence of substantially similar variant as comparedwith the polypeptide of which it is a variant are selected so as toavoid generating MHC Class I epitopes. Various methods known in the artcan be used to predict whether a sequence comprises an MHC Class Iepitope.

The structure of compstatin is known in the art, and NMR structures fora number of compstatin analogs having higher activity than compstatinare also known (Malik, supra). Structural information may be used todesign compstatin mimetics. In some embodiments, a compstatin mimetic isany compound that competes with compstatin or any compstatin analog(e.g., a compstatin analog whose sequence is set forth in Table 2) forbinding to C3 or a fragment thereof (such as a 40 kD fragment of the βchain to which compstatin binds). In some embodiments, the compstatinmimetic has an activity equal to or greater than that of compstatin. Insome embodiments, the compstatin mimetic is more stable, orallyavailable, or has a better bioavailability than compstatin. Thecompstatin mimetic may be a peptide, nucleic acid, or small molecule. Incertain embodiments the compstatin mimetic is a compound that binds tothe binding site of compstatin as determined in a compstatin-C3structure, e.g., a crystal structure or a 3-D structure derived from NMRexperiments. In certain embodiments the compstatin mimetic is a compoundthat could substitute for compstatin in a compstatin-C3 structure andwould form substantially the same intermolecular contacts with C3 ascompstatin. In certain embodiments the compstatin mimetic is a compoundthat binds to the binding site of a peptide having a sequence set forthin Table 2, e.g., SEQ ID NO: 14, 21, 28, 29, 32, 33, 34, or 36 or othercompstatin analog sequence or in certain embodiments SEQ ID NO: 30 or31, in a peptide-C3 structure. In certain embodiments the compstatinmimetic is a compound that could substitute for a peptide having asequence set forth in Table 2, e.g., SEQ ID NO: 14, 21, 28, 29, 32, 33,34, or 36 or other compstatin analog sequence or in certain embodimentsSEQ ID NO: 30 or 31, in a peptide-C3 structure and would formsubstantially the same intermolecular contacts with C3 as the peptide.In certain embodiments the compstatin mimetic has a non-peptide backbonebut has side chains arranged in a sequence designed based on thesequence of compstatin.

One of skill in the art will appreciate that once a particular desiredconformation of a short peptide has been ascertained, methods fordesigning a peptide or peptidomimetic to fit that conformation are wellknown. See, e.g., G. R. Marshall (1993), Tetrahedron, 49: 3547-3558;Hruby and Nikiforovich (1991), in Molecular Conformation and BiologicalInteractions, P. Balaram & S. Ramasehan, eds., Indian Acad. of Sci.,Bangalore, P P. 429-455), Eguchi M, Kahn M., Mini Rev Med Chem.,2(5):447-62, 2002. Of particular relevance to the present invention, thedesign of peptide analogs may be further refined by considering thecontribution of various side chains of amino acid residues, e.g., forthe effect of functional groups or for steric considerations asdescribed in the art for compstatin and analogs thereof, among others.

It will be appreciated by those of skill in the art that a peptide mimicmay serve equally well as a peptide for the purpose of providing thespecific backbone conformation and side chain functionalities requiredfor binding to C3 and inhibiting complement activation. Accordingly, itis contemplated as being within the scope of the present invention toproduce and utilize C3-binding, complement-inhibiting compounds throughthe use of either naturally-occurring amino acids, amino acidderivatives, analogs or non-amino acid molecules capable of being joinedto form the appropriate backbone conformation. A non-peptide analog, oran analog comprising peptide and non-peptide components, is sometimesreferred to herein as a “peptidomimetic” or “isosteric mimetic,” todesignate substitutions or derivations of a peptide that possesses muchthe same backbone conformational features and/or other functionalities,so as to be sufficiently similar to the exemplified peptides to inhibitcomplement activation. More generally, a compstatin mimetic is anycompound that would position pharmacophores similarly to theirpositioning in compstatin, even if the backbone differs.

The use of peptidomimetics for the development of high-affinity peptideanalogs is well known in the art. Assuming rotational constraintssimilar to those of amino acid residues within a peptide, analogscomprising non-amino acid moieties may be analyzed, and theirconformational motifs verified, by means of the Ramachandran plot (Hruby& Nikiforovich 1991), among other known techniques.

One of skill in the art will readily be able to establish suitablescreening assays to identify additional compstatin mimetics and toselect those having desired inhibitory activities. For example,compstatin or an analog thereof could be labeled (e.g., with aradioactive or fluorescent label) and contacted with C3 in the presenceof different concentrations of a test compound. The ability of the testcompound to diminish binding of the compstatin analog to C3 isevaluated. A test compound that significantly diminishes binding of thecompstatin analog to C3 is a candidate compstatin mimetic. For example,a test compound that diminishes steady-state concentration of acompstatin analog-C3 complex, or that diminishes the rate of formationof a compstatin analog-C3 complex by at least 25%, or by at least 50%,is a candidate compstatin mimetic. One of skill in the art willrecognize that a number of variations of this screening assay may beemployed. Compounds to be screened include natural products, librariesof aptamers, phage display libraries, compound libraries synthesizedusing combinatorial chemistry, etc. The invention encompassessynthesizing a combinatorial library of compounds based upon the coresequence described above and screening the library to identifycompstatin mimetics. Any of these methods could also be used to identifynew compstatin analogs having higher inhibitory activity than compstatinanalogs tested thus far.

Other Compounds that Inhibit C3 Activation or Activity

Other compounds, e.g., polypeptides, small molecules, monoclonalantibodies, aptamers, etc., that bind to C3 or C3a receptors (C3aR) areof use in certain embodiments of the invention. In certain embodimentsthe complement inhibitor comprises an Efb protein from Staphylococcusaureus or a variant or derivative or mimetic thereof that can bind to C3and inhibit its activation and/or bind to and inhibit C3b. Exemplaryagents are described in PCT Application Pub. WO/2004/094600. In certainembodiments the complement inhibitor comprises a Staphylococcuscomplement inhibitor (SCIN) protein from Staphylococcus aureus or avariant or derivative or mimetic of such protein that can bind to C3convertase and inhibit its activation and/or bind to and inhibit C3b.Aptamers that bind to and inhibit C3 may be identified using methodssuch as SELEX. U.S. Pat. Pub. No. 20030191084 discloses aptamers thatbind to C1q, C3 and C5.

In some embodiments, a protease that degrades C3 may be used as acomplement inhibitor. For example, U.S. Pat. No. 6,676,943 discloseshuman complement C3-degrading protein from Streptococcus pneumoniae.Such proteins, or variants thereof, may be used in certain embodimentsof the invention.

U.S. Pat. No. 5,942,405, PCT/IB2006/002557 (WO/2007/034277-ARYLSUBSTITUTED IMIDAZO [4,5-C] PYRIDINE COMPOUNDS AS C3A RECEPTORANTAGONISTS); PCT/IB2006/002568 (WO/2007/034282-DIARYL-IMIDAZOLECOMPOUNDS CONDENSED WITH A HETEROCYCLE AS C3A RECEPTOR ANTAGONISTS)PCT/IB2006/002561 (WO2007034278—FUSED IMIDAZOLE DERIVATIVES AS C3ARECEPTOR ANTAGONISTS) PCT/US2007/026237 (WO2008079371) MODULATORS OF C3ARECEPTOR AND METHODS OF USE THEREOF disclose exemplary C3aR antagonists.In some embodiments, an RNAi agent that inhibits expression of C3 orC3aR may be used.

Compounds that Inhibit Factor B Activation or Activity

In certain embodiments a complement inhibitor inhibits activation oractivity of factor B. For example, the complement inhibitor may bind tofactor B and, e.g., inhibit activation of factor B. Exemplary agentsthat inhibit activation or activity of factor B include, e.g.,antibodies, antibody fragments, peptides, small molecules, and aptamers.Exemplary antibodies that inhibit factor B are described in U.S. Pat.Pub. No. 20050260198. In certain embodiments an antibody orantigen-binding fragment selectively binds to factor B within the thirdshort consensus repeat (SCR) domain. In certain embodiments the antibodyprevents formation of a C3bBb complex. In certain embodiments theantibody or antigen-binding fragment prevents or inhibits cleavage offactor B by factor D. In some embodiments, an antibody binds to the Bbportion of factor B. PCT/US2008/074489 (WO/2009/029669) disclosesexemplary antibodies, e.g., the antibody produced by the hybridoma clonedeposited under ATCC Accession Number PTA-8543. In some embodiments, ahumanized version of said antibody is used, which may be an antibodyfragment. In certain embodiments a complement inhibitor, e.g., antibody,small molecule, aptamer, polypeptide, or peptide, binds to substantiallythe same binding site on factor B as an antibody described in U.S. Pat.Pub. No. 20050260198 or WO/2009/029669. In some embodiments, thecomplement inhibitor comprises the monoclonal antibody fragment known asTA106 (formerly under development by Taligen Therapeutics), or antibody,small molecule, aptamer, polypeptide, or peptide, binds to substantiallythe same binding site on factor B as TA106 is used. In some embodiments,a peptide that binds to and inhibits factor B is identified using, forexample, a method such as phage display. In some embodiments, acomplement inhibitor comprises an aptamer that binds to and inhibitsfactor B. In some embodiments, an RNAi agent that inhibits expression offactor B may be used.

Compounds that Inhibit Factor D Activity

In certain embodiments the complement inhibitor inhibits factor D. Forexample, the complement inhibitor may bind to factor D. Exemplary agentsinclude antibodies, antibody fragments, peptides, small molecules, andaptamers. Exemplary antibodies that inhibit factor D are described inU.S. Pat. No. 7,112,327. In certain embodiments the complement inhibitoris an antibody, small molecule, aptamer, or polypeptide that binds tosubstantially the same binding site on factor D as an antibody describedin U.S. Pat. No. 7,112,327. FCFD4514S (formerly under development byTanox as TNX-234), is a humanized monoclonal antibody fragment thatbinds Factor D. In certain embodiments the complement inhibitorcomprises FCFD4514S or an antibody, small molecule, aptamer, orpolypeptide that binds to substantially the same binding site on factorD as FCFD4514S. Exemplary polypeptides that inhibit alternative pathwayactivation and are believed to inhibit factor D are disclosed in U.S.Pub. No. 20040038869. Use of peptides that bind to and inhibit factor D,which may be identified using methods such as phage display, is withinthe scope of the invention. Use of aptamers that bind to and inhibitfactor D, which may be identified using methods such as SELEX, is withinthe scope of the invention. In some embodiments, an RNAi agent thatinhibits expression of factor D may be used.

Mammalian Complement Regulatory Proteins and Complement Receptors

In some embodiments the complement inhibitor comprises at least aportion of a mammalian, e.g., human, complement regulatory protein orcomplement receptor. Examples of complement regulatory proteins include,e.g., CFH, CFH related proteins (such as CFHR1), CFI, CR1, DAF, MCP,CD59, C4 bp, and complement receptor 2 inhibitor trispanning (CRIT;Inal, J., et al, J Immunol., 174(1):356-66, 2005). In some embodimentsthe complement regulatory polypeptide is one that is normallymembrane-bound in its naturally occurring state. In some embodiments ofthe invention a fragment of such polypeptide that lacks some or all of atransmembrane and/or intracellular domain is used. Soluble forms ofcomplement receptor 1 (sCR1), or soluble portions of other complementreceptors, for example, are of use in certain embodiments. For examplethe compounds known as TP10 or TP20 (Avant Therapeutics) can be used. Insome embodiments a soluble complement control protein, e.g., CFH or aCFH related protein, is used. In some embodiments the complementinhibitor is a C3b/C4b Complement Receptor-like molecule such as thosedescribed in U.S. Pat. Pub. No. 20020192758. Variants and fragments ofmammalian complement regulatory proteins or receptors that retaincomplement inhibiting activity can be used in certain embodiments.

Chimeric Complement Inhibitors

In certain embodiments of the invention the complement inhibitorcomprises a chimeric polypeptide comprising a first polypeptide thatinhibits complement activation, linked, e.g., covalently linked, to asecond polypeptide that inhibits complement activation and/or that bindsto a complement component or complement activation product. In someembodiments, at least one of the polypeptides comprises at least aportion of a mammalian complement regulatory protein. The chimericpolypeptide may contain one or more additional domains located, e.g.,between the first and second polypeptides or at a terminus. For example,the first and second polypeptides can be separated by a spacerpolypeptide.

In some embodiments, the first and second polypeptides each comprise atleast a portion of a mammalian complement regulatory protein. In someembodiments complement inhibitor comprises at least a portion of DAF andat least a portion of MCP. Exemplary chimeric polypeptides aredisclosed, e.g., in U.S. Pat. No. 5,679,546, e.g., CAB-2 (also known asMLN-2222). In some embodiments the polypeptide comprises at least 4 SCRdomains of at least one mammalian complement regulatory protein orcomplement receptor. In some embodiments the polypeptide comprises atleast 4 SCR domains of each of first and second distinct mammaliancomplement regulatory proteins.

In some embodiments, a chimeric polypeptide comprises at least a portionof complement receptor 1 (CR1), complement receptor 2 (CR2), complementreceptor 3 (CR3), complement receptor 4 (CR4) or a variant or fragmentof CR1, CR2, CR3, or CR4 that binds to one or more complement componentsor complement activation products such as C3b, iC3b, C3d, and/or C3dg.In some embodiments, the polypeptide comprises at least 4 SCRs, e.g., atleast 4 SCRs of CR1 or CR2. For example, the polypeptide can comprisethe 4 N-terminal SCRs of CR2 (e.g., residues 1-250 of the matureprotein). In some embodiments the chimeric polypeptide comprises atleast 4 SCR domains of a mammalian complement regulatory protein and atleast 4 SCR domains of a mammalian complement receptor.

Compounds that Inhibit Properdin

In some embodiments of the invention antiproperdin antibodies, antibodyfragment, or other anti-properdin agents are used. See, e.g., U.S. Pat.Pub. No. 20030198636 or PCT/US2008/068530 (WO/2009/110918-ANTI-PROPERDINANTIBODIES) for examples.

Compounds that Inhibit Components of Lectin Pathway

In some embodiments the compounds inhibit one or more components of thelectin pathway. See, e.g., WO/2007/117996) METHODS FOR TREATINGCONDITIONS ASSOCIATED WITH MASP-2 DEPENDENT COMPLEMENT ACTIVATION.

Compounds that Inhibit C5 Activation or Activity

In certain embodiments the complement inhibitor inhibits activation ofC5. For example, the complement inhibitor may bind to C5 and inhibit itscleavage. In some embodiments, the complement inhibitor inhibitsphysical interaction of C5 with C5 convertase by, e.g., binding to C5 orC5 convertase or to C5 at a site that would ordinarily participate insuch physical interaction. Exemplary agents that inhibit C5 activationinclude antibodies, antibody fragments, polypeptides, small molecules,and aptamers. Exemplary compounds, e.g., antibodies, that bind to C5 aredescribed, for example, in U.S. Pat. No. 6,534,058; PCT/US95/05688 (WO1995/029697), PCT/EP2010/007197 (WO2011063980); U.S. Pat. Pub. No.20050090448; and U.S. Pat. Pub. No. 20060115476. U.S. Pat. Pub. No.20060105980 discloses aptamers that bind to and inhibit C5. In someembodiments, a humanized anti-C5 monoclonal antibody, e.g., eculizumab(also known as h5G1.1-mAb; Soliris®) (Alexion), or a fragment orderivative thereof that binds to C5. In some embodiments, an antibodycomprising at least some of the same complementarity determining regions(CDR1, CDR2 and/or CDR3), e.g., all of CDR1, CDR2, and CDR3, as those ofeculizumab's heavy chain and/or light chain is used. In someembodiments, the antibody comprises at least some of the same frameworkregions as eculizumab. In some embodiments, an antibody that binds tosubstantially the same binding site on C5 as eculizumab is used. In someembodiments, pexelizumab (also known as h5G1.1-scFv), a humanized,recombinant, single-chain antibody derived from h5G1.1-mAb, is used. Incertain embodiments the complement inhibitor comprises a StaphylococcusSSL7 protein from Staphylococcus aureus or a variant or derivative ormimetic of such protein that can bind to C5 and inhibit its cleavage.

As noted above, bispecific or multispecific antibodies can be used. Forexample, PCT/US2010/039448 (WO/2010/151526) discloses bispecificantibodies described as binding to two or more different proteins,wherein at least two of the proteins are selected from C5a, C5b, acellular receptor for C5a (e.g., C5aR1 or C5L2), the C5b-9 complex, anda component or intermediate of terminal complement such as C5b-6, C5b-7,or C5b-8. In some embodiments an RNAi agent that inhibits expression ofC5 or C5aR may be used.

In some embodiments, a complement inhibitor known as OmCI, or a variant,derivative, or mimetic thereof, is used. OmCI binds to C5 and inhibitsits activation most likely by inhibiting interaction with convertase.OmCI is naturally produced by the tick Ornithodoros moubata. See, e.g.,PCT/GB2004/002341 (WO/2004/106369) and PCT/GB2010/000213(WO/2010/100396), for description of OmCI and certain variants thereof.It has been shown that OmCI binds to eicosanoids, in particularleukotriene (LKs), e.g., LTB4. In some embodiments, an OmCI polypeptide(or a variant, derivative, or fragment thereof) that retains thecapacity to binds to a LK, e.g., LTB4, is used. In some embodiments, anOmCI polypeptide (or a variant, derivative, or fragment thereof) thathas reduced capacity or substantially lacks capacity to bind to a LK,e.g., LTB4, is used.

In some embodiments the agent is an antagonist of a C5a receptor (C5aR).In some embodiments, the C5aR antagonist comprises a peptide. ExemplaryC5a receptor antagonists include a variety of small cyclic or acyclicpeptides such as those described in March, D R, et al., Mol. Pharmacol.,65(4), 2004, and in Woodruff, T M, et al., J Pharmacol Exp Ther.,314(2):811-7, 2005, U.S. Pat. No. 6,821,950; U.S. Ser. No. 11/375,587;and/or PCT/US06/08960 (WO2006/099330), or a mimetic thereof. In certainembodiments the complement inhibitor binds to C5aR and inhibits bindingof C5a thereto. In certain embodiments a cyclic peptide comprising thesequence [OPdChaWR] (SEQ ID NO: 59) is used. In certain embodiments acyclic peptide comprising the sequence [KPdChaWR] (SEQ ID NO: 60) isused. In certain embodiments a peptide comprising the sequence(Xaa)_(n)[OPdChaWR] (SEQ ID NO: 61) is used, wherein Xaa is an aminoacid residue and n is between 1 and 5. In certain embodiments a peptidecomprising the sequence (Xaa)_(n)[KPdChaWR] (SEQ ID NO: 62) is used,wherein Xaa is an amino acid residue and n is between 1 and 5. Incertain embodiments n is 1. In certain embodiments n is 1 and Xaa is astandard or nonstandard aromatic amino acid. For example, the peptidesF-[OPdChaWR] (SEQ ID NO: 63), F-[KPdChaWR] (SEQ ID NO: 64);Cin-[OPdChaWR] (SEQ ID NO: 65), and HCin-[OPdChaWR] (SEQ ID NO: 66) areof use in certain embodiments. Optionally the free terminus comprises ablocking moiety, e.g., the terminal amino acid is acetylated. Forexample, in some embodiments the C5aR antagonist is AcF-[OPdChaWR] (SEQID NO: 67) (also known as PMX-53). (Abbreviations: 0: ornithine; Cha:cyclohexylalanine; Cin: cinnamoyl; Hcin: hydrocinnamoyl; square bracketsdenote internal peptide bond). In some embodiments, a C5aR antagonistcomprises a compound, e.g., a peptide, disclosed in U.S. Pat. Pub. No.20060183883 (U.S. Ser. No. 10/564,788), e.g., a compound as representedtherein by formula I, formula II, formula IV, formula V, or formula VI.An exemplary C5aR antagonist is the peptide known as JPE-1375 (JeriniAG, Germany)

In some embodiments, a C5aR antagonist is a small molecule. Varioussmall molecule C5aR antagonists are disclosed in the followingreferences: PCT/US2005/015897 (WO/2005/110416; 4,5-DISUBSTITUTED-2-ARYLPYRIMIDINES); PCT/EP2006/005141 (WO2006128670); PCT/US2008/072902(WO/2009/023669; SUBSTITUTED 5,6,7,8-TETRAHYDROQUINOLINE DERIVATIVES);PCT/US2009/068941 (WO/2010/075257; C5AR ANTAGONISTS). An exemplary smallmolecule C5aR antagonist is CCX168 (ChemoCentryx, Mountain View,Calif.).

In certain embodiments the complement inhibitor is an agent, e.g., anantibody, small molecule, aptamer, or polypeptide, that binds tosubstantially the same binding site on C5 or C5aR as a compounddescribed in any of the afore-mentioned references disclosing agentsthat bind to C5 or C5aR. In some embodiments the complement inhibitor isnot an antagonist of a C5a receptor.

Multimodal Complement Inhibitors

In certain embodiments of the invention the complement inhibitor bindsto more than one complement protein and/or inhibits more than one stepin a complement activation pathway. Such complement inhibitors arereferred to herein as “multimodal”. In certain embodiments of theinvention the complement inhibitor comprises a virus complement controlprotein (VCCP). The invention contemplates use of any of the agentsdescribed in U.S. Ser. No. 11/247,886 and PCT/US2005/36547. Poxvirusesand herpes viruses are families of large, complex viruses with a lineardouble-stranded DNA genome. Certain of these viruses encodeimmunomodulatory proteins that are believed to play a role inpathogenesis by subverting one or more aspects of the normal immuneresponse and/or fostering development of a more favorable environment inthe host organism (Kotwal, G J, Immunology Today, 21(5), 242-248, 2000).Among these are VCCPs. Poxvirus complement control proteins are membersof the complement control protein (CCP) superfamily and typicallycontain 4 SCR modules. In certain embodiments the VCCP is a poxviruscomplement control protein (PVCCP). The PVCCP can comprise a sequenceencoded by, e.g., vaccinia virus, variola major virus, variola minorvirus, cowpox virus, monkeypox virus, ectromelia virus, rabbitpox virus,myxoma virus, Yaba-like disease virus, or swinepox virus. In otherembodiments the VCCP is a herpesvirus complement control protein(HVCCP). The HVCCP can comprise a sequence encoded by a Macaca fuscatarhadinovirus, cercopithecine herpesvirus 17, or human herpes virus 8. Inother embodiments the HVCCP comprises a sequence encoded by herpessimplex virus saimiri ORF 4 or ORF 15 (Albrecht, J C. & Fleckenstein,B., J. Virol., 66, 3937-3940, 1992; Albrecht, J., et al., Virology, 190,527-530, 1992).

The VCCP may inhibit the classical complement pathway, the alternatecomplement pathway, the lectin pathway, or any two or more of these. Incertain embodiments of the invention the VCCP, e.g., a PVCCP, binds toC3b, C4b, or both. In certain embodiments of the invention the PVCCPcomprises one or more putative heparin binding sites (K/R-X-K/R) and/orpossesses an overall positive charge. In some embodiments the PVCCPcomprises at least 3 SCR modules (e.g., modules 1-3), e.g., 4 SCRmodules. The PVCCP protein can be a precursor of a mature PVCCP (i.e.,can include a signal sequence that is normally cleaved off when theprotein is expressed in virus-infected cells) or can be a mature form(i.e., lacking the signal sequence).

Vaccinia complement control protein (VCP) is a virus-encoded proteinsecreted from vaccinia infected cells. VCP is 244 amino acids in length,contains 4 SCRs, and is naturally produced by intracellular cleavage ofa 263 amino acid precursor. VCP runs as an ˜35 kD protein in a 12%SDS/polyacrylamide gel under reducing conditions and has a predictedmolecular mass of about 28.6 kD. VCP is described in U.S. Pat. Nos.5,157,110 and 6,140,472, and in Kotwal, G K, et al., Nature, 355,176-178, 1988. FIGS. 3A and 3B of U.S. Ser. No. 11/247,886 andPCT/US2005/36547 (WO2006042252) show the sequence of the precursor andmature VCP proteins, respectively. VCP has been shown to inhibit theclassical pathway of complement activation via its ability to bind to C3and C4 and act as a cofactor for factor I mediated cleavage of thesecomponents as well as promoting decay of existing convertase (Kotwal, GK, et al., Science, 250, 827-830, 1990; McKenzie et al., J. Infect.Dis., 1566, 1245-1250, 1992). It has also been shown to inhibit thealternative pathway by causing cleavage of C3b into iC3b and therebypreventing the formation of the alternative pathway C3 convertase (Sahu,A, et al., J. Immunol., 160, 5596-5604, 1998). VCP thus blockscomplement activation at multiple steps and reduces levels of theproinflammatory chemotactic factors C3a, C4a, and C5a.

VCP also possesses the ability to strongly bind heparin in addition toheparan sulfate proteoglycans. VCP contains two putative heparin bindingsites located in modules 1 and 4 (Jha, P and Kotwal, G J, and referencestherein). VCP is able to bind to the surface of endothelial cells,possibly via interaction with heparin and/or heparan sulfate at the cellsurface, resulting in decreased antibody binding (Smith, S A, et al., J.Virol., 74(12), 5659-5666, 2000). VCP can be taken up by mast cells andpossibly persist in tissue for lengthy periods of time, therebypotentially prolonging its activity (Kotwal, G J, et al., In GP. Talwat,et al. (eds), 10^(th) International Congress of Immunology., MonduzziEditore, Bologna, Italy, 1998). In addition, VCP can reduce chemotacticmigration of leukocytes by blocking chemokine binding (Reynolds, D, etal., in S. Jameel and L. Villareal (ed., Advances in animal virology.Oxford and IBN Publishing, New Delhi, India, 1999). VCP and other PVCCPshave a relatively small size relative to mammalian CCPs, which isadvantageous for delivery in the present invention.

Variola virus major and minor encode proteins that are highly homologousto VCP and are referred to as smallpox inhibitor of complement enzymes(SPICE) (Rosengard, A M, et al., Proc. Natl. Acad. Sci., 99(13),8803-8813. U.S. Pat. No. 6,551,595). SPICE from various variola strainssequenced to date differs from VCP by about 5% (e.g., about 11 aminoacid differences). Similarly to VCP, SPICE binds to C3b and C4b andcauses their degradation, acting as a cofactor for factor I. However,SPICE degrades C3b approximately 100 times as fast as VCP and degradesC4b approximately 6 times as fast as VCP. The amino acid sequence ofSPICE is presented in FIG. 6 (SEQ ID NO: 12) of U.S. Ser. No. 11/247,886and PCT/US2005/36547 (WO2006042252) and can be described as follows.Referring to FIG. 6 of U.S. Ser. No. 11/247,886 and PCT/US2005/36547(WO2006042252), a signal sequence extends from amino acid 1 to aboutamino acid 19. Four SCRs extend from about amino acid 20 to amino acid263. Each SCR is characterized by four cysteine residues. The fourcysteine residues form two disulfide bonds in the expressed protein. Theboundaries of each SCR are best defined by the first and fourth cysteineresidues in the sequence that forms the disulfide bonds of the SCR. Aninvariant tryptophan residue is present between cysteine 3 and cysteine4 of each SCR. SCR1 extends from amino acid 20 or 21 to amino acid 81.Both residues are cysteines that may be involved in disulfide bonding.SCR2 extends from amino acid 86 to amino acid 143. SCR3 extends fromamino acid 148 to amino acid 201. SCR4 extends from amino acid 206 toamino acid 261. The SCRs include the complement binding locations ofSPICE. SPICE or any of the portions thereof that inhibit complementactivation, e.g., SPICE and SPICE-related polypeptides containing fourSCRs, such as those described in U.S. Pat. No. 6,551,595, are of use inthe present invention.

Complement control proteins from cowpox virus (referred to asinflammation modulatory protein, IMP) and monkeypox virus (referred toherein as monkeypox virus complement control protein, MCP) have alsobeen identified and sequenced (Miller, C G, et al., Virology, 229,126-133, 1997 and Uvarova, E A and Shchelkunov, S N, Virus Res.,81(1-2), 39-45, 2001). MCP differs from the other PVCCPs describedherein in that it contains a truncation of the C-terminal portion of thefourth SCR.

It will be appreciated that the exact sequence of complement controlproteins identified in different virus isolates may differ slightly.Such proteins fall within the scope of the present invention. Complementcontrol proteins from any such isolate may be used, provided that theprotein has not undergone a mutation that substantially abolishes itsactivity. Thus the sequence of a VCCP such as SPICE or VCP may differfrom the exact sequences presented herein or under the accession numberslisted in Table 3. It will also be appreciated that a number of aminoacid alterations, e.g., additions, deletions, or substitutions such asconservative amino acid substitutions, may be made in a typicalpolypeptide such as a VCCP without significantly affecting its activity,such that the resulting protein is considered equivalent to the originalpolypeptide. The viral polypeptides identified by accession number inTable 3 below are of use in various embodiments of the invention.

TABLE 3 Representative Viral Complement Control Proteins Virus ProteinAccession Virus Type Variola D12L NP_042056 Orthopoxvirus D15L (SPICE)AAA69423 Orthopoxvirus Vaccinia VCP AAO89304 Orthopoxvirus CowpoxCPXV034 AAM13481 Orthopoxvirus C17L CAA64102 Orthopoxvirus MonkeypoxD14L AAV84857 Orthopoxvirus Ectromelia virus Complement control proteinCAE00484 Orthopoxvirus Rabbitpox RPXV017 AAS49730 Orthopoxvirus Macacafuscata rhadinovirus JM4 AAS99981 Rhadinavirus (Herpesvirus)Cercopithecine herpesvirus 17 Complement binding NP_570746 Herpesvirusprotein (ORF4) Human herpes virus 8 Complement binding AAB62602Herpesvirus protein (ORF4)

In addition to the VCCPs described above, a number of other viralproteins exist that interfere with one or more steps in a complementpathway. These proteins are also of use in certain embodiments of thepresent invention. Certain of these proteins do not necessarily displayclear homology to cellular complement regulators known to date. Forexample, HSV-1, HSV-2, VZV, PRV, BHV-1, EHV-1, and EHV-4 all encodeversions of a conserved glycoprotein known as gC (Schreurs, et al., JVirol., 62, 2251-2257, 1988; Mettenleiter, et al, J. Virol., 64,278-286; 1990; Herold, et al., J Virol., 65, 1090-1098; 1991). With theexception of VZV, the gC protein encoded by these viruses binds to C3b(Friedman, et al., Nature, 309, 633-634,1984; Huemer, et al., VirusRes., 23, 271-280, 1993) gC1 (from HSV-1) accelerates decay of theclassical pathway C3 convertase and inhibits binding of properdin and C5to C3. Purified EBV virions possess an activity that accelerates decayof the alternative pathway C3 convertase and serves as a cofactor forthe complement regulatory protein factor 1 (Mold et al., J Exp Med, 168,949-969, 1988). The foregoing proteins are referred to collectively asvirus complement interfering proteins (VCIPs). By any of a variety ofmeans, such as interfering with one or more steps of complementactivation, accelerating decay of a complement component, and/orenhancing activity of a complement regulatory protein, these VCIPs aresaid to inhibit complement. Any of these proteins, or derivativesthereof, e.g., fragments or variants thereof, can be used as atherapeutic agent in the invention. As in the case of VCCPs, will beappreciated that the exact sequence of VCIPs identified in differentvirus isolates may differ slightly. Such proteins fall within the scopeof the present invention.

In certain embodiments of the invention a fragment or variant of a VCCPor VCIP is locally administered to a subject. Preferred fragments andvariants of a PVCCP possess at least one of the following activities:(i) ability to bind to C3, C3b, or both; (ii) ability to act as acofactor for factor I cleavage of C3; (iii) ability to bind to C4, C4b,or both; (iv) ability to act as a cofactor for factor I cleavage of C4;(v) ability to accelerate decay of existing C3 convertase of theclassical pathway, alternate pathway, or both; (vi) ability to bindheparin; (vii) ability to bind to heparan sulfate proteoglycans; (viii)ability to reduce chemotactic migration of leukocytes; (ix) ability toblock chemokine (e.g, MIP-1α) binding, e.g., to the surface of a cell(e.g., a leukocyte or endothelial cell surface); (x) ability to inhibitantibody binding to class I MHC molecules; (xi) ability to inhibit theclassical complement pathway; (xii) ability to inhibit the alternativecomplement pathway; and (xiii) ability to inhibit complement-mediatedcell lysis. Preferred PVCCP fragments and variants display complementbinding activity, by which is meant ability to detectably bind to one ormore complement components, preferably (in the case of VCCPs) selectedfrom the group consisting of: C3, C3b, C4, and C4b. Preferred fragmentsor variants of HVCCPs may also display ability to detectably bind to oneor more complement components. Preferably the binding of the VCCP to thecomplement component is specific. It will be understood that a VCCP maybe able to bind to only a single complement component or may be able tobind to more than one different complement component.

In certain embodiments of the invention the PVCCP fragment or variantcomprises at least 3 SCR modules (e.g., modules 1-3), preferably 4 SCRmodules. Preferably each of the SCR modules displays significantsequence identity to an SCR module found in a naturally occurring PVCCP,e.g., VCP or SPICE. Preferably the multiple SCR modules are arranged inan N to C manner so as to maximize overall identity to a naturallyoccurring PVCCP. If the sequence of a PVCCP fragment or variant containsan SCR domain that differs from the SCR consensus sequence at one ormore positions, in certain embodiments of the invention the aminoacid(s) at the one or more differing positions is identical to thatfound at a corresponding position in the most closely related SCR foundin a naturally occurring PVCCP. In certain embodiments the PVCCP variantcomprises at least one SCR module from a first PVCPP and at least oneSCR module from a second PVCPP. In certain embodiments the PVCCP variantcomprises at least one SCR module from a PVCCP and at least one SCR froma mammalian complement control protein (RCA protein). Any number of SCRmodules, e.g., 1, 2, 3, 4, or more can come from any particular PVCCP orRCA protein in various embodiments of the invention. All suchcombinations and permutations are contemplated, even if not explicitlyset forth herein.

Generally a fragment or variant of a naturally occurring VCCP or VCIPpossesses sufficient structural similarity to its naturally occurringcounterpart that it is recognized by a polyclonal antibody thatrecognizes the naturally occurring counterpart. In certain embodimentsof the invention a fragment or variant of a VCCP possesses sufficientstructural similarity to VCP or SPICE so that when its 3-dimensionalstructure (either actual or predicted structure) is superimposed on thestructure of VCP or SPICE, the volume of overlap is at least 70%,preferably at least 80%, more preferably at least 90% of the totalvolume of the VCP structure. A partial or complete 3-dimensionalstructure of the fragment or variant may be determined by crystallizingthe protein as described for VCP (Murthy, 2001). Alternately, an NMRsolution structure can be generated, as performed for various VCPfragments (Wiles, A P, et al., J. Mol. Biol. 272, 253-265, 1997). Amodeling program such as MODELER (Sali, A. and Blundell, T L, J. Mol.Biol., 234, 779-815, 1993), or any other modeling program, can be usedto generate a predicted structure. The model can be based on the VCPstructure and/or any known SCR structure. The PROSPECT-PSPP suite ofprograms can be used (Guo, J T, et al., Nucleic Acids Res. 32(Web Serverissue):W522-5, Jul. 1, 2004). Similar methods may be used to generate astructure for SPICE.

Fragments or variants of a VCCP or VCIP may be generated by anyavailable means, a large number of which are known in the art. Forexample, VCCPs, VCIPs, and fragments or variants thereof can be producedusing recombinant DNA technology as described below. A VCCP or VCIPfragment may be chemically synthesized, produced using PCR amplificationfrom a cloned VCCP or VCIP sequence, generated by a restriction digest,etc. Sequences for a VCCP variant may be generated by random mutagenesisof a VCCP sequence (e.g., using X-rays, chemical agents, or PCR-basedmutagenesis), site-directed mutagenesis (e.g., using PCR oroligonucleotide-directed mutagenesis, etc. Selected amino acids can bechanged or added.

While not wishing to be bound by any theory, it is likely that aminoacid differences between naturally occurring PVCCPs occur at positionsthat are relevant in conferring differences in particular propertiessuch as ability to bind heparin, activity level, etc. For example, VCPand SPICE differ at only 11 amino acids, but SPICE has a much higheractivity as a cofactor for cleavage of C3b (e.g., cleavage occurs at amuch faster rate with SPICE than with VCP). The amino acid differencesare likely to be responsible for the differential activities of the twoproteins. The amino acids at these positions are attractive candidatesfor alteration to identify variants that have yet greater activity.

Additional Complement Inhibitors

In some embodiments a complement inhibitor is a naturally occurringmammalian complement regulatory protein or a fragment or derivativethereof. For example, the complement regulatory protein may be CR1, DAF,MCP, CFH, or CFI. In some embodiments of the invention the complementregulatory polypeptide is one that is normally membrane-bound in itsnaturally occurring state. In some embodiments of the invention afragment of such polypeptide that lacks some or all of a transmembraneand/or intracellular domain is used. Soluble forms of complementreceptor 1 (sCR1), for example, are of use in the invention. For examplethe compounds known as TP10 or TP20 (Avant Therapeutics) can be used. C1inhibitor (C1-INH) is also of use. In some embodiments a solublecomplement control protein, e.g., CFH, is used. In some embodiments ofthe invention the polypeptide is modified to increase its solubility.

In some embodiments, a complement inhibitor is a C1s inhibitor. Forexample, U.S. Pat. No. 6,515,002 describes compounds (furanyl andthienyl amidines, heterocyclic amidines, and guanidines) that inhibitC1s. U.S. Pat. Nos. 6,515,002 and 7,138,530 describe heterocyclicamidines that inhibit C1s. U.S. Pat. No. 7,049,282 describes peptidesthat inhibit classical pathway activation. Certain of the peptidescomprise or consist of WESNGQPENN (SEQ ID NO: 68) or KTISKAKGQPREPQVYT(SEQ ID NO: 69) or a peptide having significant sequence identity and/orthree-dimensional structural similarity thereto. In some embodimentsthese peptides are identical or substantially identical to a portion ofan IgG or IgM molecule. U.S. Pat. No. 7,041,796 discloses C3b/C4bComplement Receptor-like molecules and uses thereof to inhibitcomplement activation. U.S. Pat. No. 6,998,468 discloses anti-C2/C2ainhibitors of complement activation. U.S. Pat. No. 6,676,943 discloseshuman complement C3-degrading protein from Streptococcus pneumoniae.

V. Anti-Th17 Agents

An anti-Th17 agent is any agent that inhibits formation, survival,and/or activity of Th17 cells or that inhibits production or abiological activity of a Th17 cell effector molecule such as IL-17. Insome embodiments an anti-Th17 agent inhibits development, proliferation,survival, and/or maturation of Th17 cells. In some embodiments, ananti-Th17 agent inhibits production and/or a biological activity ofIL-6, IL-21, IL-23, and/or IL-1β. In some embodiments, an anti-Th17agent inhibits production and/or activity of a Th17 effector cytokine,e.g., IL-17A, IL-17F, IL-21, and/or IL-22. Exemplary anti-Th17 agentsinclude, e.g., agents that bind to a Th17-associated cytokine or agentsthat bind to a receptor for a Th17-associated cytokine and, e.g., blockinteraction of the receptor with the endogenous cytokine but do notthemselves significantly activate the receptor. Exemplary anti-Th17agents include, e.g., antibodies, aptamers, soluble receptor fragments(e.g., soluble extracellular domain of the relevant cytokine receptor)or other polypeptides, peptides, small molecules, etc. In someembodiments, an anti-Th17 agent comprises an antibody that substantiallylacks the capacity to activate complement. For example, the antibody mayhave less than 10%, less than 5%, or less than 1% complement stimulatingactivity as compared with full length human IgG1. In some embodiments,the antibody comprises a CH2 domain that has reduced ability to bind C1qas compared with human IgG1 CH2 domain. In some embodiments, theantibody contains CH1, CH2, and/or CH3 domains from human IgG4 and/ordoes not contain CH1, CH2, and/or CH3 domains from human IgG1.

In some embodiments, an anti-Th17 agent has a molecular weight of 1 kDor less. In some embodiments, an anti-Th17 agent has a molecular weightbetween 1 kD and 2 kD, between 2 kD and 5 kD, between 5 kD and 10 kD,between 10 kD and 20 kD, between 20 kD and 30 kD, between 30 kD and 50kD, between 50 kD and 100 kD, or between 100 kD and 200 kD.

In some embodiments an anti-Th17 agent comprises an adnectin, affibody,anticalin, or other type of polypeptide sometimes used in the art inlieu of an antibody, wherein the polypeptide binds to a Th17-associatedcytokine or cytokine receptor.

A variety of anti-Th17 agents, e.g., agents that inhibit one or moreTh17-associated cytokines, are known in the art and may be used invarious embodiments.

Sequences of polypeptides of interest herein, e.g., Th17-associatedcytokines and their receptors, are well known in the art and availablein public databases such as those available through Entrez at theNational Center for Biotechnology Information website or UniversalProtein Resource website. Exemplary databases include, e.g., GenBank,RefSeq, Gene, Protein, Nucleotide, UniProtKB/SwissProt,UniProtKB/Trembl, and the like. In general, sequences, e.g., mRNA andpolypeptide sequences, in the NCBI Reference Sequence database may beused as gene product sequences for a gene of interest. Such sequencesmay be used, e.g., to produce a polypeptide useful as an antigen orreagent for production, isolation, or characterization of an agent thatbinds to the gene product. It will be appreciated that multiple allelesof a gene may exist among individuals of the same species. For example,differences in one or more nucleotides (e.g., up to about 1%, 2%, 3-5%of the nucleotides) of the nucleic acids encoding a particular proteinmay exist among individuals of a given species. Due to the degeneracy ofthe genetic code, such variations often do not alter the encoded aminoacid sequence, although DNA polymorphisms that lead to changes in thesequence of the encoded proteins can exist. Examples of polymorphicvariants can be found in, e.g., the Single Nucleotide PolymorphismDatabase (dbSNP), available at the NCBI website. (Sherry S T, et al.(2001). “dbSNP: the NCBI database of genetic variation”. Nucleic AcidsRes. 29 (1): 308-311; Kitts A, and Sherry S, (2009). The singlenucleotide polymorphism database (dbSNP) of nucleotide sequencevariation in The NCBI Handbook [Internet]. McEntyre J, Ostell J,editors. Bethesda (Md.): National Center for Biotechnology Information(US); 2002. Multiple isoforms of certain proteins may exist, e.g., as aresult of alternative RNA splicing or editing. In general, where aspectsof this disclosure pertain to a gene or gene product, embodimentspertaining to allelic variants or isoforms are encompassed unlessindicated otherwise. Certain embodiments may be directed to particularsequence(s), e.g., particular allele(s) or isoform(s).

Table 4 lists Gene ID and NCBI RefSeq accession numbers for certainhuman Th17-associated cytokines and their receptors. It will beappreciated that certain of the protein sequences are precursorsequences. The mature form of the protein would lack a secretion signalsequence present in the precursor. It will be appreciated that thesequences described under the respective accession numbers for thecytokines and cytokine receptors listed in Table 4 are exemplary andthat naturally occurring variants, e.g., allelic variants, areencompassed in various embodiments. Furthermore, it will be appreciatedthat for purposes of generating a useful binding agent (e.g., anantibody) for use as a detection reagent or therapeutic agent, variantsequences, short peptide segments, etc., may be used in certainembodiments.

TABLE 4 Gene ID and Accession Numbers for Certain Th17-AssociatedCytokines and their Receptors Protein and mRNA mRNA Protein GeneOfficial Alternate name Gene accession accession Symbol/Name andcomments ID number number IL23A/interleukin p19 51561 NM_016584NP_057668 23, alpha subunit IL12B/interleukin p40; IL23 beta 3593NM_002187 NP_002178 12B subunit. IL23R/interleukin alpha subunit 149233NM_144701 NP_653302 23 receptor of the IL23 receptor IL12RB/interleukinbeta subunit 3594 NM_005535 NP_005526 12 receptor, beta 1 of the IL23(isoform 1) (isoform 1) receptor NM_153701 NP_714912 (isoform 2)(isoform 2) IL17A 3605 NM_002190 NP_002181 IL17F 112744 NM_052872NP_443104 IL-17RA/ 23765 NM_014339 NP_055154 interleukin 17 receptor A

In some embodiments an anti-Th17 agent is an anti-IL-23 agent. An IL-23agent is an agent (e.g., a molecule or complex) that partially or fullybocks, inhibits, neutralizes, prevents or interferes with a biologicalactivity of IL-23. In some embodiments a biological activity of IL-23 isthe ability to induce IL-17 production by activated T cells. IL-23 is aheterodimeric cytokine composed of two subunits. The IL-23 beta subunit,also called p40, is shared with another cytokine, interleukin-12(IL-12). The IL-23 alpha subunit is also called p19. The IL-23 subunitsare joined by a disulfide bond. IL-23 signals via binding to aheterodimeric receptor, composed of IL-12Rbetal (IL12RB1), which isshared by the IL-12 receptor, and IL-23R (Parham C, et al. (2002) J.Immunol. 168 (11): 5699-708). IL-23R associates constitutively withJanus kinase 2 (JAK2), and also binds to transcription activator STAT3in a ligand-dependent manner. The IL-23 signal transduction cascadeparallels those of various other cytokines, in that ligand binding leadsto activation of JAKs. The JAKs then phosphorylate the IL-23R at keysites, forming docking sites for the STATs. Subsequently, the JAKsphosphorylate the STATs, which dimerize and translocate to the nucleuswhere they activate target genes. In some embodiments an anti-IL-23agent comprises an antibody that binds to the p19 or p40 subunit ofIL-23. In some embodiments an anti-IL-23 agent, e.g., an anti-IL-23antibody, binds to the p40 subunit and inhibits both IL-23 and IL-12.

Certain anti-IL-23 agents and methods of identifying and/or making suchagents are disclosed in U.S. Ser. No. 10/697,599. For example, screeningmethods and assays that may be readily employed by the ordinary skilledartisan to identify and/or produce a variety of anti-IL-23 agents(referred to sometimes as “IL-23 antagonists” in U.S. Ser. No.10/697,599) are disclosed.

In certain embodiments an anti-IL-23 antibody that binds to the p40subunit of IL-23 is ustekinumab or a fragment thereof. Ustekinumab(experimental name CNTO 1275, proprietary commercial name Stelara®,Centocor; CAS Number: 815610-63-0) is a human monoclonal antibody of theIgG1 subclass. Exemplary anti-IL-23 antibodies that bind to the p19subunit of human IL-23, and isolated nucleic acids that encode at leastone anti-IL-23p19 antibody, vectors, host cells, and methods of making,are described in U.S. Ser. No. 11/617,503. Additional anti-IL-23antibodies that bind to the p19 subunit are described in U.S. Ser. No.11/762,738.

In some embodiments an anti-IL-23 agent comprises an IL-23p40 specificimmunoglobulin derived proteis (see, e.g., U.S. Ser. No. 11/768,582).

In some embodiments an IL-23 inhibitor comprises a polypeptidecomprising a soluble IL-23R or a variant or fragment thereof capable ofbinding to IL-23 in solution. In some embodiments a soluble IL-23R lacksthe portion of IL-23R encoded by exon 9 of the IL-23R alpha gene. See,e.g., Yu, R Y, J Immunol. (2010) 15; 185(12):7302-8. In some embodimentsa soluble IL-23R lacks the portion of IL-23 encoded by exon 9 and atleast a portion of exon 8 of the IL-23R alpha gene.

In some embodiments, IL-23 activity is inhibited by interfering withIL-23 signal transduction, e.g., by inhibiting one or more processes orproteins involved in the IL-23 signal transduction pathway. For example,in some embodiments IL-23 signaling is inhibited using a JAK inhibitoror a STAT inhibitor. In some embodiments a JAK inhibitor inhibits JAKexpression. Methods of use to inhibit JAK expression in some embodimentsinclude the use of RNAi agents (e.g., siRNA) or antisenseoligonucleotides. In some embodiments a JAK inhibitor inhibits JAKbinding to IL-23 receptor. In some embodiments a JAK inhibitor inhibitsJAK dimerization. In some embodiments a JAK inhibitor inhibits JAKkinase activity. For example, in some embodiments a JAK inhibitor bindsto the JAK kinase domain, e.g., to the ATP binding site. Numerous JAKinhibitors are known in the art. For example, INCB028050 is an orallybioavailable JAK1/JAK2 inhibitor with reported nanomolar potency againstJAK1 (5.9 nM) and JAK2 (5.7 nM) (Fridman, J S, et al., J Immunol. 2010;184(9):5298-307). INCB028050 is reported to inhibit intracellularsignaling of multiple proinflammatory cytokines including IL-6 and IL-23at concentrations <50 nM. Small molecule JAK2 inhibitors include, e.g.,AZD1480 and AZ960.

In some embodiments a STAT inhibitor inhibits STAT expression. Methodsof use to inhibit STAT expression in some embodiments include the use ofRNAi agents (e.g., siRNA) or antisense oligonucleotides. In someembodiments a STAT inhibitor inhibits STAT binding to JAK. In someembodiments a STAT inhibitor inhibits STAT dimerization or nucleartranslocation. In some embodiments a STAT inhibitor comprises aphosphopeptide which, e.g., competes with STAT for binding tophosphorylated JAK. WO/2008/151037 discloses certain peptide-based STATinhibitors of use in certain embodiments. In some embodiments a STATinhibitor inhibits STAT binding to DNA. For example, a decoyoligonucleotide comprising a sequence substantially identical to anendogenous DNA sequence to which STAT naturally binds in human cells maybind to STAT and prevent it from binding to its endogenous bindingsite(s). Small molecule STAT3 inhibitors include, e.g., STA-21, IS3 295,and S3I-M2001. See Huang, S., Clin Cancer Res 2007; 13:1362-1366 andreferences therein, which are incorporated herein by reference, forfurther information regarding certain STAT inhibitors.

In some embodiments an anti-Th17 agent is an anti-IL-17 agent. An IL-17agent is an agent (e.g., a molecule or complex) that partially or fullybocks, inhibits, neutralizes, prevents or interferes with a biologicalactivity of IL-17. Exemplary anti-IL-17 polypeptides, e.g., anti-IL-17antibodies, are described in, e.g., U.S. Ser. No. 11/658,344. Additionalanti-IL-17 antibodies are described in U.S. Ser. No. 11/762,738. In someembodiments an anti-IL-17 agent comprises at least a portion of an IL-17receptor, wherein the portion binds to IL-17. Exemplary IL-17 receptorpolypeptides are disclosed in, e.g., U.S. Ser. No. 09/022,260.

It will be understood that a polypeptide comprising a binding domain ofany of the various anti-Th17 antibodies or other polypeptides describedherein can be transferred into other polypeptide backbones or used asisolated agents in certain embodiments. It will further be understoodthat variants may be used. For example, a variant may be at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to abinding domain of a receptor. In some embodiments an antibody thatcompetes with a particular antibody known in the art for binding to acytokine of interest may be used. In some embodiments an antibody of theIgG class is modified so that it lacks an Fc domain that may activatecomplement. For example, a variable domain of an IgG1 antibody may begrafted to a constant region of an IgG4 antibody.

VI. Pharmaceutical Compositions and Administration Approaches

Suitable preparations, e.g., substantially pure preparations of acomplement inhibitor may be combined with pharmaceutically acceptablecarriers or vehicles, etc., to produce an appropriate pharmaceuticalcomposition. The term “pharmaceutically acceptable carrier or vehicle”refers to a non-toxic carrier or vehicle that does not destroy thepharmacological activity of the compound with which it is formulated.One of skill in the art will understand that a carrier or vehicle is“non-toxic” if it is compatible with administration to a subject in anamount appropriate to deliver the compound without causing unduetoxicity. Pharmaceutically acceptable carriers or vehicles that may beused include, but are not limited to, water, physiological saline,Ringer's solution, sodium acetate or potassium acetate solution, 5%dextrose, and the like. The composition may include other components asappropriate for the formulation desired, e.g., as discussed herein.Supplementary active compounds, e.g., compounds independently useful fortreating a subject suffering from a respiratory disorder, can also beincorporated into the compositions. The invention provides suchpharmaceutical compositions comprising a complement inhibitor and,optionally, a second active agent useful for treating a subjectsuffering from a respiratory disorder.

In some embodiments, the invention provides a pharmaceuticallyacceptable complement inhibitor or pharmaceutically acceptablecomposition comprising a complement inhibitor, packaged together with apackage insert (label) approved by a government agency responsible forregulating pharmaceutical agents, e.g., the U.S. Food & DrugAdministration. In some embodiments, the invention provides apharmaceutical pack comprising: (a) a pharmaceutically acceptablecomplement inhibitor in concentrated or solid form (e.g., as alyophilized powder); (b) a pharmaceutically acceptable carrier, diluent,or vehicle. In some embodiments, a carrier, diluent, or vehicle issuitable for use to deliver the composition using a nebulizer. In someembodiments, a suitable carrier, diluent, or vehicle may be providedseparately or acquired by a health care provider from an appropriatesource. Optionally a pack contains instructions for dissolving ordiluting the complement inhibitor in the carrier, diluent, or vehicle toproduce a composition for administration. In some embodiments a packageinsert states one or more indications that include one or more chroniccomplement-mediated disorders, e.g., one or more chronic respiratorydisorders, e.g., asthma or COPD. In some embodiments, the package insertstates particular patient and/or disease characteristics or criteriathat define a patient population or disease category for treatment ofwhich the composition has been approved for use. In some embodiments,the package insert specifies that the composition may be or should beadministered according to a method of the present invention, e.g.,according to a dosing schedule and/or using a dosing interval describedherein.

In general, a pharmaceutical composition can be administered to asubject by any suitable route of administration including, but notlimited to, intravenous, intramuscular, subcutaneously, by therespiratory route, etc. In some embodiments, local administration to atissue or organ affected by a complement-mediated disorder is used. Itwill be understood that “administration” encompasses directlyadministering a compound or composition to a subject, instructing athird party to administer a compound or composition to a subject,prescribing or suggesting a compound or composition to a subject (e.g.,for self-administration), self-administration, and, as appropriate,other means of making a compound or composition available to a subject.If administration is accomplished using an implanted reservoir,administration can refer to causing release of a composition or compoundfrom the reservoir.

Pharmaceutical compositions suitable for injectable use (e.g.,intravenous administration, subcutaneous or intramuscularadministration) typically include sterile aqueous solutions (where watersoluble) or dispersions and sterile powders for the extemporaneouspreparation of sterile injectable solutions or dispersion. Sterilesolutions can be prepared by incorporating the compound in the requiredamount in an appropriate solvent, optionally with one or a combinationof ingredients such as buffers such as acetates, citrates, lactates orphosphates; agents for the adjustment of tonicity such as sodiumchloride or dextrose; antibacterial agents such as benzyl alcohol ormethyl parabens; antioxidants such as ascorbic acid, glutathione, orsodium bisulfate; chelating agents such as ethylenediaminetetraaceticacid; and other suitable ingredients etc., as desired, followed byfilter-based sterilization. One of skill in the art will be aware ofnumerous physiologically acceptable compounds that may be included in apharmaceutical composition. Other useful compounds include, for example,carbohydrates, such as glucose, sucrose, lactose; dextrans; amino acidssuch as glycine; polyols such as mannitol. These compounds may, forexample, serve as bulking agents and/or stabilizers, e.g., in a powderand/or when part of the manufacture or storage process involveslyophilization. Surfactant(s) such as Tween-80, Pluronic-F108/F68,deoxycholic acid, phosphatidylcholine, etc., may be included in acomposition, e.g., to increase solubility or to provide microemulsion todeliver hydrophobic drugs. pH can be adjusted with acids or bases, suchas hydrochloric acid or sodium hydroxide, if desired. The parenteralpreparation can be enclosed in ampoules, disposable syringes or infusionbags or multiple dose vials made of glass or plastic. Preferablysolutions for injection are sterile and acceptably free of endotoxin.

Generally, dispersions are prepared by incorporating the active compoundinto a sterile vehicle which contains a basic dispersion medium andappropriate other ingredients from those enumerated above. In the caseof sterile powders for the preparation of sterile injectable solutions,methods of preparation can include vacuum drying and freeze-drying whichyields a powder of the active ingredient plus any additional desiredingredient, e.g., from a previously sterile-filtered solution thereof.

For administration by the respiratory route (inhalation), a complementinhibitor may be delivered in the form of an aerosol spray from apressured container or dispenser which contains a suitable propellant. Ametered dose inhaler (MDI), dry powder inhaler, or nebulizer may beused. The aerosol may comprise liquid and/or dry particles (e.g., drypowders, large porous particles, etc.). Suitable aqueous vehicles usefulin various embodiments include water or saline, optionally including analcohol. In some embodiments the composition comprises a surfactantsuitable for introduction into the lung. Other excipients suitable forpulmonary administration can be used.

A variety of different devices are available for respiratoryadministration. Nebulizers are devices that transform solutions orsuspensions of medications into aerosols that are suitable fordeposition in the lower airway. Nebulizer types include jet nebulizers,ultrasonic wave nebulizers, and vibrating mesh nebulizers. A partiallist of available vibrating mesh nebulizers includes eFlow (Pari), i-Neb(Respironics), MicroAir (Omron), IH50 Nebulizer (Beurer), and Aeroneb®(Aerogen). A Respimat® Soft Mist™ Inhaler (Boeringer Ingelheim) may beused. A metered dose inhaler (MDI) is a handheld aerosol device thatuses a propellant to deliver the therapeutic agent. MDIs include apressurized metal canister that contains pharmacological agent insuspension or solution, propellant, surfactant (typically), and meteringvalve. Chloroflourocarbons (CFCs) had been widely used as propellantsbut have been largely replaced by hydrofluorocarbons (HFCs, also knownas hydrofluoroalkanes (HFA)) such as HFC-134a and HFC-227ea. Carbondioxide and nitrogen are other alternatives. A dry powder inhaler (DPI)is a breath-actuated device that delivers the drug in the form ofparticles contained in a capsule or blister that is punctured prior touse and typically does not employ a propellant. Examples of DPIscurrently used to deliver medications for treating asthma and/or COPDinclude, e.g., Diskus, Aerolizer, HandiHaler, Twisthaler, Flexhaler.Such devices may be used to deliver a complement inhibitor in variousembodiments of the invention. Other exemplary DPI devices that may beused in various embodiments include 3M™ Taper and 3M Conix™, TAIFUN®(AKELA Pharma), Acu-Breathe™ (Respirics).

Inhalation accessory devices (IADs) generally fall into 2 categories:spacers and holding chambers. Spacers and holding chambers extend themouthpiece of the inhaler and direct the mist of medication toward themouth, reducing medication lost into the air. Using a spacer with an MDIcan help reduce the amount of drug that sticks to the back of thethroat, improving direction and deposition of medication delivered byMDIs. Valved holding chambers (VHCs) allow for a fine cloud ofmedication to stay in the spacer until the patient breathes it inthrough a one-way valve, drawing the dose of medicine into the lungs.Examples include Aerochamber and Optichamber.

Particulate compositions may be characterized on the basis of variousparameters such as the fine particle fraction (FPF), the emitted dose,the average particle density, and the mass median aerodynamic diameter(MMAD). Suitable methods are known in the art, some of which aredescribed in U.S. Pat. Nos. 6,942,868 and 7,048,908 and U.S. PublicationNos. 20020146373, 20030012742, and 20040092470. In certain embodimentsaerosol particles are between approximately 0.5 μm-10 μm (MMAD), e.g.,about 5 μm for respiratory delivery, though smaller or larger particlescould also be used. In certain embodiments particles having a mass meanaerodynamic diameter of between 1 μm and 25 μm, e.g., between 1 μm and10 μm, are used.

A dry particle composition containing particles smaller than about 1 mmin diameter is also referred to herein as a dry powder. A “dry”composition has a relatively low liquid content, so that the particlesare readily dispersible, e.g., in a dry powder inhalation device to forman aerosol or spray. A “powder” consists largely or essentially entirelyof finely dispersed solid particles that are relatively free flowing andcapable of being readily dispersed in an inhalation device andsubsequently inhaled by a subject, preferably so that a significantfraction of the particles can reach a desired portion of the respiratorytract. In certain embodiments large porous particles having meangeometric diameters ranging between 3 and 15 μm and tap density between0.04 and 0.6 g/cm³ are used. See, e.g., U.S. Pat. No. 7,048,908;Edwards, D. et al, Science 276:1868-1871, 1997; and Vanbever, R., etal., Pharmaceutical Res. 16:1735-1742, 1999).

Various considerations for respiratory delivery that may be useful inembodiments of the present invention are discussed in Bisgaard, H., etal., (eds.), Drug Delivery to the Lung, Vol. 26 in “Lung Biology inHealth and Disease”, Marcel Dekker, New York, 2002. Aerosol devices arediscussed, e.g., in Dolovich M B, Dhand R. Lancet. (2011)377(9770):1032-45.

Oral administration may be used in certain embodiments. Oralcompositions generally include an inert diluent or an edible carrier.For the purpose of oral therapeutic administration, the active compoundcan be incorporated with excipients and used in the form of tablets,troches, or capsules, e.g., gelatin capsules. Pharmaceuticallycompatible binding agents, and/or adjuvant materials can be included aspart of the composition. The tablets, pills, capsules, troches and thelike can contain any of the following ingredients, or compounds of asimilar nature: a binder such as microcrystalline cellulose, gumtragacanth or gelatin; an excipient such as starch or lactose, adisintegrating agent such as alginic acid, Primogel, or corn starch; alubricant such as magnesium stearate or Sterotes; a glidant such ascolloidal silicon dioxide; a sweetening agent such as sucrose orsaccharin; or a flavoring agent such as peppermint, methyl salicylate,or orange flavoring. A liquid composition can also be administeredorally. Formulations for oral delivery may incorporate agents to improvestability within the gastrointestinal tract and/or to enhanceabsorption.

For topical application, a complement inhibitor may be formulated in asuitable ointment containing the active component suspended or dissolvedin one or more carriers. Carriers for topical administration include,but are not limited to, mineral oil, liquid petrolatum, whitepetrolatum, propylene glycol, polyoxyethylene, polyoxypropylenecompound, emulsifying wax and water. Alternatively, the pharmaceuticallyacceptable compositions can be formulated as a suitable lotion or creamcontaining a compstatin analog suspended or dissolved in one or morepharmaceutically acceptable carriers. Suitable carriers include, but arenot limited to, mineral oil, sorbitan monostearate, polysorbate 60,cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol,and water.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated may be used in theformulation. Such penetrants are generally known in the art, andinclude, for example, for transmucosal administration, detergents, bilesalts, and fusidic acid derivatives. Transmucosal administration can beaccomplished, e.g., through the use of nasal sprays or suppositories. Insome embodiments, intranasal administration is used, e.g., to administera complement inhibitor to a subject in need of treatment for nasalpolyposis, chronic rhinosinusitis, or allergic rhinitis. For transdermaladministration, the active compounds are typically formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

Methods of local administration to the eye include, e.g., intraocularadministration, e.g., intraocular injection, e.g., intravitrealinjection. In some embodiments, administration is by choroidalinjection, transscleral injection, eyedrops or eye ointments,transretinal, subconjunctival bulbar, intravitreal injection,suprachoroidal injection, subtenon injection, scleral pocket or scleralcutdown injection.

In certain embodiments of the invention, a complement inhibitor isprepared with carrier(s) that will protect the compound against rapidelimination from the body, such as a controlled release formulation,including implants and microencapsulated delivery systems. For example,a compound may be incorporated into or encapsulated in a microparticleor nanoparticle formulation. Biodegradable, biocompatible polymers canbe used, such as ethylene vinyl acetate, polyanhydrides, polyglycolicacid, collagen, polyorthoesters, polyethers, polylactic acid, PLGA, etc.Liposomes or other lipid-based particles can be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811 and/or other references listed herein. Depot formulationscontaining a complement inhibitor may be used. The complement inhibitoris released from the depot over time. One of ordinary skill in the artwill appreciate that the materials and methods selected for preparationof a controlled release formulation, implant, etc., should be such as toretain activity of the compound. In some embodiments, a composition isfree or essentially free of one or more carrier(s) whose primary or onlyintended purpose or effect would be to result in sustained or controlledrelease of an active agent, e.g., a complement inhibitor.

In some embodiments, a complement inhibitor is used in combination withone or more additional active agent(s) useful to treat a disorder ofinterest herein (see, e.g., Brunton, L L, et al. (eds.), Goodman andGilman's The Pharmacological Basis of Therapeutics, (e.g., 11th or 12thedition), McGraw-Hill, for examples of such agents.) in some embodimentsone or more additional active agents is administered in the samecomposition as a complement inhibitor. In some embodiments one or moreadditional active agents is administered in a separate composition,which separate composition may be administered prior to, atapproximately the same time as, or after administration of a complementinhibitor. In some embodiments, use of a complement inhibitor allowsreduction in dose and/or frequency of administration of an additionalactive agent while maintaining at least equivalent disease controland/or benefit to the subject. It will be understood that pharmaceuticalcompositions comprising an additional active agent may be prepared usingpharmaceutically acceptable carriers and/or preparation methodsdescribed herein or known in the art, and administered using routes ofadministration described herein or known in the art.

In some embodiments a second active agent is an agent that interfereswith the DC-Th17-B-Ab-C-DC cycle by a mechanism distinct from directinhibition of a complement component or complement activation. In someembodiments a second active agent may be an anti-IL-23 agent oranti-IL-17 agent. In some embodiments a pharmaceutical composition orpharmaceutical pack comprises a second active agent that interferes withthe DC-Th17-B-Ab-C-DC cycle. In some embodiments a package insertspecifies that two agents are to be administered in combination. In someembodiments a complement inhibitor, e.g., a compstatin analog, may beadded to any treatment regimen that comprises an anti-Th17 agent. Insome embodiments such addition permits a lower dose or increased dosinginterval of the anti-Th17 agent to be used, without reduction inefficacy. In some embodiments such addition results in increasedefficacy.

When two or more therapies (e.g., compounds or compositions) are used oradministered “in combination” with each other, they may be given at thesame time, within overlapping time periods, or sequentially (e.g.,separated by up to 2-4 weeks in time), in various embodiments of theinvention. They may be administered via the same route or differentroutes in various embodiments. They may be administered in either orderin various embodiments. In some embodiments, the compounds orcompositions are administered within 4, 8, 12, 24, 48, 72, or 96 hoursof each other. In some embodiments, a first agent is administered priorto or after administration of the second agent, e.g., sufficiently closein time that the two agents are present at useful levels within the bodyat least once. In some embodiments, the agents are administeredsufficiently close together in time such that no more than 90% of theearlier administered composition has been metabolized to inactivemetabolites or eliminated, e.g., excreted, from the body, at the timethe second compound or composition is administered. In some embodiments,the agents are administered sufficiently close together in time suchthat no more than 2 weeks has elapsed since the earlier administeredagent has been metabolized to inactive metabolites or eliminated, e.g.,excreted, from the body, at the time the second agent is administered.In some embodiments administration of two agents (e.g., a complementinhibitor and a second agent that interferes with the DC-Th17-B-Ab-C-DCcycle act additively, resulting in an effect that would not be achievedby either agent alone. In some embodiments administration of two agents(e.g., a complement inhibitor and a second agent that interferes withthe DC-Th17-B-Ab-C-DC cycle act synergistically, resulting in an effectthat is greater than an additive effect and/or is qualitativelydifferent to an additive effect in a clinically and/or statisticallysignificant way.

It will be appreciated that a complement inhibitor and/or additionalactive agent(s) can be provided as a pharmaceutically acceptable salt.Pharmaceutically acceptable salts include those derived frompharmaceutically acceptable inorganic and organic acids and bases.Examples of suitable acid salts include acetate, adipate, alginate,aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate,camphorate, camphorsulfonate, cyclopentanepropionate, digluconate,dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate,glycerophosphate, glycolate, hemisulfate, heptanoate, hexanoate,hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate,lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate,nicotinate, nitrate, oxalate, palmoate, pectinate, persulfate,3-phenylpropionate, phosphate, picrate, pivalate, propionate,salicylate, succinate, sulfate, tartrate, thiocyanate, tosylate andundecanoate. Also, pharmaceutically-acceptable salts can be prepared asalkaline metal or alkaline earth salts, such as sodium, potassium orcalcium salts, if appropriate depending on the identity of the activeagent.

It will be understood that the pharmaceutically acceptable carriers,compounds, and preparation methods mentioned herein are exemplary andnon-limiting. See, e.g., Remington: The Science and Practice ofPharmacy. 21st Edition. Philadelphia, Pa. Lippincott Williams & Wilkins,2005, for additional discussion of pharmaceutically acceptable compoundsand methods of preparing pharmaceutical compositions of various types.

A compound or composition, e.g., a pharmaceutical composition, can beused or administered to a subject in an effective amount. In someembodiments, an “effective amount” of an active agent, e.g., acomplement inhibitor, (or composition containing an active agent) refersto an amount of the active agent (or composition) sufficient to elicitone or more biological response(s) of interest in, for example, asubject to whom the active agent (or composition) is administered. Aswill be appreciated by those of ordinary skill in the art, the absoluteamount of a particular agent that is effective may vary depending onsuch factors as the biological endpoint, the particular active agent,the target tissue, etc. Those of ordinary skill in the art will furtherunderstand that an “effective amount” may be administered in a singledose, or may be achieved by administration of multiple doses. Forexample, in some embodiments, an effective amount may be an amountsufficient to achieve one or more of the following: (i) reduce theseverity of one or more manifestations (e.g., one or more symptoms orsigns) of a chronic respiratory disorder; (ii) cause a reduction infrequency and/or severity of exacerbations (which reduction may resultin, e.g., decreased days of school or work lost, decreased physicianand/or emergency room visits, decreased hospitalization events, and/ordecreased mortality); (iii) permit a reduction in use of standardmedication for the disorder while maintaining at least equivalentdisease control; and/or (iv) inhibit or prevent a long-term pathologicalchange associated with the disorder; and/or (v) improve daily function.In many embodiments, a therapeutically relevant effective amount atleast in part reduces one or more manifestations (e.g., symptoms) of achronic disorder and/or returns one or more physiological or biochemicalparameters or indicators associated with or causative of a chronicdisorder at least partially to normal. For example, in some embodiments,an effective amount may be an amount sufficient to achieve one or moreof the following: (i) reduce the severity of one or more manifestations(e.g., one or more symptoms or signs) of a chronic respiratory disorder;(ii) reduce the magnitude of EAR, LAR, and/or DAR (as assessed, forexample, by maximum reduction in FEV₁ and/or maximum reduction in PEFmeasured within a relevant time period following an allergen challenge);(iii) reduce likelihood of developing an EAR, LAR, and/or DAR; (iv)cause a reduction in frequency and/or severity of exacerbations (whichreduction may result in, e.g., decreased days of school or work lost,decreased physician and/or emergency room visits, decreasedhospitalization events, and/or decreased mortality); (v) permit areduction in use of ICS, OCS, leukotriene modifiers, and/or Xolair whilemaintaining at least equivalent disease control; (vi) inhibit or preventairway remodeling; (vii) improve daily function and/or exercisetolerance; and/or (viii) reduce one or more indicators of airwayinflammation. In many embodiments in which an agent is administered to asubject in need of treatment for a chronic respiratory disorder, atherapeutically relevant effective amount at least in part reduces oneor more manifestations (e.g., symptoms) of a chronic respiratorydisorder and/or returns one or more physiological or biochemicalparameters or indicators associated with or causative of a chronicrespiratory disorder at least partially to normal.

Indicators of airway inflammation include, e.g., the presence ofincreased numbers of inflammation-associated cells such as white bloodcells (e.g., eosinophils, lymphocytes, macrophages, and/or neutrophils)and/or inflammatory mediators (e.g., chemokines (e.g., eotaxin, thymusand activation-regulated chemokine (TARC), macrophage-derived chemokine(MDC)), cytokines (e.g., TNFalpha, IL-1beta, IL-4, IL-5, IL-13, IL-25),histamine, cysteinyl leukotrienes, nitric oxide) in the airways, ascompared with a suitable reference level, e.g., a normal level. Forexample, the number and/or concentration of cells and/or mediators maybe above the upper limit of the normal range in subjects not sufferingfrom a disorder (where “normal range” typically refers to a range ofwithin ±2 standard deviations from a mean value in a population ofsubjects) or may be greater than a value (or average value) measured inthat subject when the subject's disorder is well controlled. A reductionin symptom severity and/or frequency can be statistically significantand/or clinically meaningful within the sound judgment of a physician orother medical practitioner. Determining whether a disorder is wellcontrolled is within the sound judgment of a physician or other medicalpractitioner. Art-accepted guidelines may be used.

In some embodiments an effective amount results in reduction of at leastone parameter associated with Th17 cells and/or Th17 activity. In someembodiments an effective amount reduces the level of at least onecytokine associated with Th17 cells and/or Th17 activity, e.g., acytokine that promotes Th17 cell formation and/or activity or a cytokineproduced by Th17 cells. In some embodiments a cytokine is IL-17, IL21,IL-22, or IL-23. In some embodiments an effective amount results in ashift from Th17 to Treg cells. In some embodiments a shift from Th17cells to Treg cells is reflected in an immune micro-environment that isrelatively rich in IL-10 and relatively poor in IL-17 and IL-23.

For treatment of AMD, an effective amount may be an amount sufficient toachieve one or more of the following: (i) inhibit or prevent drusenformation; (ii) cause a reduction in drusen number and/or size (drusenregression); (iii) cause a reduction in or prevent lipofuscin deposits;(iv) inhibit or prevent visual loss or slow the rate of visual loss; (v)inhibit choroidal neovascularization or slow the rate of choroidalneovascularization; (vi) cause a reduction in size and/or number oflesions characterized by choroidal neovascularization; (vii) inhibitchoroidal neovascularization or slow the rate of retinalneovascularization; (viii) cause a reduction in size and/or number oflesions characterized by retinal neovascularization; (ix) improve visualacuity and/or contrast sensitivity; (x) inhibit or prevent photoreceptoror RPE cell atrophy or apoptosis, or reduce the rate of photoreceptor orRPE cell atrophy or apoptosis; (xi) inhibit or prevent progression ofnon-exudative macular degeneration to exudative macular degeneration;(xii) reduce one or more indicia of inflammation, e.g., the presence ofinflammation-associated cells such as white blood cells (e.g.,neutrophils, macrophages) in the eye, the presence of endogenousinflammatory mediators, one or more symptoms such as eye pain, redness,light sensitivity, blurred vision and floaters, etc.

One of skill in the art will be aware of appropriate methods to assessthe afore-mentioned biological effects and other biological effects ofinterest. Symptoms can be assessed using standardized instruments (e.g.,questionnaires) known in the art. Any of a variety of differenthealth-related quality of life (HRQOL) instruments can be used, whichcan be generic or specifically associated with the respiratory system(e.g., asthma and/or COPD-specific). Pulmonary function tests,particularly spirometry, can be used to measure parameters of lungfunction that are frequently altered in subjects with chronicrespiratory disorders, such as FEV₁, FVC, FEV₁/FVC, PEF, etc. Allergenchallenge can be performed, e.g., as described in Kelly M M. J AllergyClin Immunol. 125(2):349-356, 2010 or studies described in Cockcroft, DW, et al. Can Respir J. 14(7): 414-418,2007. Myofibroblasts synthesizecollagen and are believed to play an important role in airway remodelingin disorders characterized by chronic airway inflammation such as asthmaand COPD. These cells are increased in the airways of asthmaticindividuals 24 h after allergen challenge. Inhibition of the increase inairway wall myofibroblasts that would otherwise occur following allergenchallenge may indicate decreased airway remodeling potential.Alternately or additionally, features associated with airway remodelingsuch as smooth muscle hyperplasia, goblet cell hyperplasia, and/orsubepithelial collagen deposition can be assessed.

Bronchial hyperreactivity can be assessed using, for example, “direct”and “indirect” challenge tests, which refer to the mode of action of theagents in relation to smooth muscle contraction. Methacholine chlorideand histamine diphosphate are most commonly used as direct smooth musclestimuli. The most frequently used indirect stimuli are hypertonicsaline, adenosine monophosphate (AMP), and mannitol. Challenge testingcan be performed, e.g., according to guidelines published by the ERS(Sterk P J, et al. Airway responsiveness. Standardized challenge testingwith pharmacological, physical and sensitizing stimuli in adults. ReportWorking Party Standardization of Lung Function Tests, European Communityfor Steel and Coal. Official Statement of the European RespiratorySociety. Eur Respir J Suppl 1993; 16:53-83) and ATS (Crapo, R O, et al.,Guidelines for methacholine and exercise challenge testing-1999. Am JRespir Crit Care Med 2000; 161:309-329). Two suitable methods forinhaling aqueous solutions of pharmacologic stimuli that may be used arethe 2-minute tidal breathing method and the dosimeter method.Bronchoconstriction causes increased airway resistance. PC(X) (where Xis a number, typically between 10 and 100) refers to the amount ofstimulus required to cause a decrease of X % in airway resistance. Ingeneral, persons with bronchial hyperreactivity, exhibit a decreasedPC(X) than normal individuals. For example individuals with bronchialhyperreactivity may have a methacholine PC(20)<4 mg/ml, whileindividuals with bronchial hyperactivity may have a PC(20) >4 mg/ml. Insome embodiments, an effective amount of a therapeutic agent increasesPC(X) in subjects suffering from a chronic respiratory disordercharacterized by bronchial hyperreactivity relative to control subjects.

Inflammation-associated cells and/or mediators may be assessed, forexample, in a suitable sample such as induced sputum, BAL fluid, and/orairway tissue sample (e.g., obtained from a biopsy such as anendobronchial biopsy). Cells, e.g., inflammation-associated cells can bedetected and optionally quantified using, e.g., electron microscopy,optical microscopy (optionally using suitable chemical stains orantibodies to particular markers (immunohistochemistry), flow cytometry,or other suitable methods. Mediator (e.g., cytokine) levels may bemeasured using, e.g., antibody-based assays such as ELISA assays, beadarray assays (such as the Luminex xMAP technology or Cytometric BeadArray (CBA) system from BD Biosciences), antibody array assays, orappropriate bioassays. Expression of mediators can alternately oradditionally be assessed by measuring the level of mRNA encoding suchmediators (e.g., using any suitable method for measuring RNA level suchas reverse transcription PCR, hybridization to oligonucleotide or cDNAarrays, RNA-Seq (e.g., methods making use of high-throughput sequencingtechnologies to sequence cDNA to obtain information about RNA in asample), etc.).

Exercise tolerance may be assessed, e.g., by testing performance on a 6minute walk test (e.g., wherein improved exercise tolerance is evidencedby an increase in the distance a subject is able to walk in 6 minutes),shuttle walk test, and/or cardiopulmonary exercise testing. See, e.g.,ATS Statement: Guidelines for the Six-Minute Walk Test (2002) fordiscussion of 6 minute walk test.

In general, a control subject can be, e.g., an untreated subject or asubject treated with a placebo. An “untreated subject” may be a subjectwho has not received treatment with a complement inhibitor within thepreceding 6 months. In some embodiments, an untreated subject has notreceived treatment with an ICS, OCS, LTRA, and/or LABA within at leastthe preceding 4 weeks. In some embodiments, an untreated subject has notreceived treatment with an anti IgE agent within at least the preceding12 weeks. Historical control information can be used. In someembodiments, a subject can serve as his or her own control. For example,one or more parameters can be measured once or more prior to treatmentand once or more during and/or following treatment. In some embodiments,an “active control” (or “active comparator”) is used, wherein abiological effect of the complement inhibitor is compared with that of acompound known to affect the parameter being assessed. For example, acompound that is approved for use as a controller medication in asthmamay be used. It will be appreciated that if an active comparator is usedas a control, an effective amount of a complement inhibitor may haveless, more, or about the same effect as the active comparator at one ormore time points in various embodiments.

In some embodiments, one or more biological effect(s) of a complementinhibitor is evident when tested at multiple time points during a dosinginterval of the instant invention, wherein said time points encompass atleast 75% of the dosing interval, e.g., at least 80%, 85%, 90%, 95%, ormore of the dosing interval. In some embodiments, one or more biologicaleffect(s) of a complement inhibitor is evident when tested at or nearthe end of the dosing interval, where “near the end of the dosinginterval” means up to 2 days before the end of the dosing interval,e.g., on the day before the end of the dosing interval.

In some embodiments, an animal model is used, for example, to help guideselection of a dose, dose range, or formulation for testing in human, toassess one or more biological effect(s), etc. Commonly used animalmodels for airway inflammation and/or asthma involve inhalation ofAscaris suum antigen. For example, inhalation of Ascaris suum antigen byallergic monkeys (e.g., cynomolgus monkey; Macaca fascicularis) causesan early bronchoconstriction and delayed allergic reaction, including apulmonary inflammatory infiltrate. See, e.g., Mellado, M., et al., JPharmacol Exp Ther. (2008) 324(2):769-75; Zou, J., et al. Genome Biol.2002; 3(5):research0020. Epub 2002 Apr. 11. Similar models exist inmice, sheep, guinea pigs, etc. In some embodiments, a significantreduction in allergen-induced EAR, LAR, and/or AHR (e.g., as assessedusing methacholine challenge) and/or a significant increase in PC(X), intreated animals as compared with untreated animals, indicateseffectiveness. In some embodiments, a reduction in EAR, LAR, and/or AHRremains evident at the end of a dosing interval selected according tothe instant invention (e.g., immediately prior to the next dose).

In general, appropriate doses of complement inhibitor or other activeagent depend at least in part upon the potency of the complementinhibitor or other active agent, route of administration, etc. Ingeneral, dose ranges that are effective and well tolerated can beselected by one of ordinary skill in the art, Such doses can bedetermined using clinical trials as known in the art. Optionally, a dosemay be tailored to the particular recipient, for example, throughadministration of increasing doses until a preselected desired responseis achieved, such as a preselected desired degree of complementinhibition and/or preselected desired reduction in response to allergenchallenge, reduction in bronchial hyperreactivity, and/or reduction inone or more symptoms of the disorder. If desired, the specific doselevel for any particular subject may be selected based at least in partupon a variety of factors including the activity of the specificcompound employed, the particular condition being treated and/or itsseverity, the age, body weight, general health, route of administration,any concurrent medication, and/or the degree of complement proteinexpression or activity measured in one or more samples obtained from thesubject. In some embodiments an effective amount or dose ranges fromabout 0.001 to 500 mg/kg body weight, e.g., about 0.01 to 100 mg/kg bodyweight, e.g., about 0.1 to 50 mg/kg body about 0.1 to 20 mg/kg bodyweight, e.g., about 1 to 10 mg/kg.

EXAMPLE 1 Effect of a Potent Compstatin Analog in Ascaris suum AnimalModel of Asthma

A potent compstatin analog having the amino acid sequence of thecompstatin analog of SEQ ID NO: 28, was synthesized using standardmethods. Briefly, amino acids were obtained as Fmoc-protected aminoacids, in which the α-amino group of each amino acid was protected withFmoc. Side chain functional groups were also blocked with variousappropriate protective groups. Synthesis was accomplished following thesolid phase methodology described by Merrifield (J. Amer. Chem. Soc. 85,2149 (1963)). Chain assembly was performed on solid phase, at theconclusion of which the N-terminus was acetylated; the peptide was thencleaved from the solid phase and simultaneously deprotected viaacidolysis using TFA and amidated. The linear peptide was then oxidizedand purified. A study designed to evaluate the efficacy of CA-28 after14 days of administration in a non-human primate model of asthma wasperformed. In this study, a dose of 15 mg/kg of CA-28 in a 2.0% glycerolsolution was administered to anesthetized animals (cynomolgus monkeys)via intratracheal nebulization once a day for 14 consecutive days usinga pneumatic nebulizer (Pari LC Plus, Pari USA, Midlothian, Va.).Budesonide (10 mg/kg, administered once daily for 9 days as a powderusing an insufflator), a glucocorticoid used for treatment of asthma,was used as a positive control. Primary endpoints included the effectson bronchoalveolar lavage (BAL) cell counts, cytokine levels, and acutepulmonary function changes as assessed by airway resistance (RL) anddynamic compliance (CDYN) after challenges with Ascaris suum (A. suum).

Animals were subjected to challenge with A. suum at 3 time points (priorto initial dose—Challenge), on day 14 (Challenge 1, i.e., the last dayof dosing), and on day 30 (Challenge 2). While each animal wasanesthetized, a single dose of A. suum antigen was administered viaintermittent positive pressure breathing with a ventilator and in-linenebulizer over 15 breaths. Each animal was administered an optimumresponse dose (ORD) which is the dose of antigen (dilution) that hashistorically elicited a >40% increase in lung resistance (R_(L)) anda >35% decrease in dynamic compliance (C_(DYN)). Blood was collected byvenipuncture and analyzed for routine clinical chemistry and hematologyparameters.

Broncheoalveolar lavage (BAL) was performed by guiding a pediatricfiberoptic bronchoscope past the carina to wedge in a major bronchus. Anattempt was made to lavage different lung fields at each time point.Three washes of sterile saline (20 mL each) were instilled andimmediately aspirated for collection into tubes. The first washcollection was placed into one 50 mL conical tube while the second andthird wash collections were combined into a second 50 mL conical tube.The samples were placed on wet ice or in a refrigerator set to maintain4° C. until transport. The cell pellets from the different washcombinations (1st/2nd/3rd wash) were combined and analyzed for total anddifferential cell counts. From stained slides, BAL cell morphology anddifferential were determined by counting a minimum of 200 nucleatedcells from all washes (cell pellets were combined from all washes), ifavailable, if less than 200 nucleated cells were available this isdocumented in the study records and results. Relative and absolutecounts were determined for macrophages, eosinophils, neutrophils,lymphocytes, and mast cells. Erythrocytes, ciliated respiratory cells,and squamous epithelial cells were not counted. BAL samples wereanalyzed for eotaxin, RANTES, IL-4, IL-5, IL-6, IL-8, IL-10, IL-13,IL-17a, IL-23, and INF-γ using qualified methods.

Results

Following aerosol A. suum antigen challenge during Challenge 0 (controlchallenge prior to dosing), all animals exhibited a severebronchoconstrictor response, which was associated with increases in lungresistance (RL) and decreases in dynamic compliance (CDYN) followed bypulmonary eosinophilia.

CA-28 did not affect the acute phase bronchoconstriction resulting fromA. suum challenge at either Challenge 2 (on the last day of CA-28dosing) or Challenge 2 (28 days after the cessation of dosing).

CA-28 resulted in slight, improvement (reduction) in eosinophiliafollowing Challenges 1 and 2. However, eosinophil counts were higher inthe baseline samples collected right after dosing and prior to the firstA. suum challenge.

Treatment with inhaled CA-28 at 15 mg/kg in a vehicle comprised of 2.0%glycerol in water resulted in lower levels of most upregulated cytokinesand chemokines as compared to animals treated with vehicle control inthe treatment session, in a way that was comparable to Budesonide inmany case, most notably eotaxin, IFN-γ, IL-4, IL-13, and IL-23, althoughthe suppression did not reach statistical significance in most cases,due to the intrinsic high variability of the data and the low number ofanimals. The most remarkable data was the total suppression of IL-23 inCA-28 treated animals observed at all time points following Challenges 1and 2. Inhibition of most of the other cytokines appeared to be presenteven following Challenge 2 in CA-28 treated groups. CA-28 upregulatedbaseline levels of IL-10, a key regulatory cytokines, following bothChallenges 1 and 2. Data are presented in graphical format in FIGS.1-11.

The data are consistent with the conclusion that CA-28 creates aprotective immune micro-environment (high IL-10, lowIFNγ/IL-4/IL-13/IL-17/IL-23) both when the drug is present in the lung(Challenge 1) and 27 days following washout of the drug (Challenge 2)(assuming a 1 day washout for both CA-28 and Budesonide). In particular,IL-17 and 11-23 levels in CA-28 treated animals 24 hours followingChallenge 2 were lower than those in control animals, suggesting asustained beneficial effect. In the case of Budesonide the IL-17/IL-23axis appears to be upregulated 24 hours following Challenge 2.

*****

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The scope of the presentinvention is not intended to be limited to the above Description, butrather is as set forth in the appended claims. It will be appreciatedthat the invention is in no way dependent upon particular resultsachieved in any specific example or with any specific embodiment.Articles such as “a”, “an” and “the” may mean one or more than oneunless indicated to the contrary or otherwise evident from the context.Claims or descriptions that include “or” between one or more members ofa group are considered satisfied if one, more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process unless indicated to the contrary or otherwiseevident from the context. The invention includes embodiments in whichexactly one member of the group is present in, employed in, or otherwiserelevant to a given product or process. For example, and withoutlimitation, it is understood that where claims or description indicatethat a residue at a particular position may be selected from aparticular group of amino acids or amino acid analogs, the inventionincludes individual embodiments in which the residue at that position isany of the listed amino acids or amino acid analogs. The invention alsoincludes embodiments in which more than one, or all of the group membersare present in, employed in, or otherwise relevant to a given product orprocess. Furthermore, it is to be understood that the inventionencompasses all variations, combinations, and permutations in which oneor more limitations, elements, clauses, descriptive terms, etc., fromone or more of the listed claims or from the description above isintroduced into another claim. For example, any claim that is dependenton another claim can be modified to include one or more elements,limitations, clauses, or descriptive terms, found in any other claimthat is dependent on the same base claim. Furthermore, where the claimsrecite a composition, it is to be understood that methods ofadministering the composition according to any of the methods disclosedherein, and methods of using the composition for any of the purposesdisclosed herein are included within the scope of the invention, andmethods of making the composition according to any of the methods ofmaking disclosed herein are included within the scope of the invention,unless otherwise indicated or unless it would be evident to one ofordinary skill in the art that a contradiction or inconsistency wouldarise. Methods of treating a subject can include a step of providing asubject in need of such treatment (e.g., a subject who has had, or is atincreased risk of having, a disease), a step of diagnosing a subject ashaving a disease and/or a step of selecting a subject for treatment witha complement inhibitor and/or anti-Th17 agent. In some embodiments amethod of treatment comprises monitoring a subject for a Th17 biomarker.In some embodiments a method of treatment comprises monitoring a subjectfor a Th17 biomarker and retreating the subject based at least in parton the result of such monitoring, e.g., administering a complementinhibitor to the subject if the biomarker indicates a resurgence of Th17cells and/or Th17-associated activity.

Where elements are presented as lists, it is to be understood that eachsubgroup of the elements is also disclosed, and any element(s) can beremoved from the group. For purposes of conciseness only some of theseembodiments have been specifically recited herein, but the inventionincludes all such embodiments. It should also be understood that, ingeneral, where the invention, or aspects or embodiments of theinvention, is/are referred to as comprising particular elements,features, etc., certain embodiments of the invention or aspects of theinvention consist, or consist essentially of, such elements, features,etc.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or subrangewithin the stated ranges in different embodiments of the invention, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise. Any embodiment, aspect, element,feature, etc., of the present invention may be explicitly excluded fromthe claims. For example, any complement inhibitor, anti-Th117 agent,carrier, formulation, formulation component, disorder, subjectpopulation or characteristic(s), dosing interval, administration route,or combination thereof can be explicitly excluded.

We claim:
 1. A method of treating a subject in need of treatment for achronic respiratory disorder or other chronic complement-mediateddisorder, the method comprising administering multiple doses of acomplement inhibitor to the subject according to a dosing schedule inwhich successive doses are administered by the intravenous, oral, orsubcutaneous route on average (i) at least 2 weeks after the plasmaconcentration of the complement inhibitor decreases to no more than 20%of the maximum plasma concentration that was reached after the previousdose; (ii) at least 2 weeks after plasma complement activation capacityhas returned to at least 50% of baseline after the previous dose; or(iii) at intervals at least equal to 3 times the terminal plasmahalf-life of the complement inhibitor when administered by the sameroute.
 2. The method of claim 1, wherein successive doses of thecomplement inhibitor are administered on average (i) between 2 weeks and6 weeks after the plasma concentration of the complement inhibitordecreases to no more than 20% of the maximum plasma concentration thatwas reached after the previous dose; or (ii) between 2 weeks and 6 weeksafter plasma complement activation capacity has returned to at least 50%of baseline after the previous dose.
 3. The method of claim 1, whereinsuccessive doses of the complement inhibitor are administered on averageat least 2 weeks after the plasma concentration of the complementinhibitor decreases to no more than 20% of the maximum plasmaconcentration that was reached after the previous dose.
 4. The method ofclaim 1, wherein at least 5 doses are administered.
 5. The method ofclaim 1., wherein the subject is in need of treatment for asthma,chronic obstructive pulmonary disease (COPD), or both.
 6. The method ofclaim 1, wherein the complement inhibitor comprises an antibody,aptamer, peptide, polypeptide, or small molecule that binds to C3, C5,factor B, or factor D.
 7. The method of claim 1, wherein the complementinhibitor comprises a compstatin analog.
 8. The method of claim 1,wherein the complement-mediated disorder is a Th17-associated disorder.9. The method of claim 1 comprising detecting a Th17 biomarker in thesubject or in a sample obtained from the subject.
 10. The method ofclaim 9, wherein the Th17 biomarker is detected in a sample comprising abody fluid.
 11. The method of claim 9, wherein the biomarker comprisesat least one cytokine that is produced by or promotes formation,survival, or activity of Th17 cells.
 12. The method of claim 9, whereinthe Th17 biomarker is detected prior to administration of a dose of thecomplement inhibitor and serves as an indicator that the subject is inneed of a dose of the complement inhibitor.
 13. The method of claim 9,wherein the biomarker is detected prior to administration of a dose ofthe complement inhibitor and serves as an indicator that the subject isin need of a dose of the complement inhibitor, and the method comprisesadministering the complement inhibitor within a 4 week time periodfollowing detection of the biomarker.
 14. A method of treating a subjectin need of treatment for a chronic complement-mediated disorder, themethod comprising: (a) administering at least one dose of a complementinhibitor to the subject according to the method of claim 1; and (b)monitoring the subject for a Th17 biomarker in the subject or in asample obtained from the subject.
 15. The method of claim 14, whereinstep (b) comprises detecting an increased level of the biomarker ascompared to a reference, wherein the increased level indicates that thesubject is in need of a dose of the complement inhibitor, and the methodfurther comprises (c) administering at least one additional dose of thecomplement inhibitor to the subject.
 16. A method of treating a subjecthaving or at risk of a Th17-associated disorder, the method comprisingmonitoring the subject for evidence of a DC-Th17-B-Ab-C-DC cycle andadministering a complement inhibitor to the subject according to adosing schedule in which successive doses are administered by theintravenous, oral, or subcutaneous route on average (i) at least 2 weeksafter the plasma concentration of the complement inhibitor decreases tono more than 20% of the maximum plasma concentration that was reachedafter the previous dose; (ii) at least 2 weeks after plasma complementactivation capacity has returned to at least 50% of baseline after theprevious dose; or (iii) at intervals at least equal to 3 times theterminal plasma half-life of the complement inhibitor when administeredby the same route based at least in part on a result of said monitoring.17. The method of claim 16, wherein the complement inhibitor inhibits C3activity or C3 activation.
 18. The method of claim 16 further comprisingadministering an anti-Th17 agent to the subject, wherein the anti-Th17agent comprises an antibody, small molecule, aptamer, or polypeptidethat binds to IL-1β, IL-6, IL-21, IL-22,IL-17, or IL-23 or binds toreceptor for any of the foregoing.
 19. The method of claim 16, whereinmonitoring the subject for evidence of a DC-Th17-B-Ab-C-DC cyclecomprises assessing a Th17-associated biomarker in the subject or in asample obtained from the subject.
 20. The method of claim 10, whereinthe body fluid is blood, BAL fluid, sputum, nasal secretion, or urine ora combination thereof.
 21. A method of treating a subject in need oftreatment for a chronic respiratory disorder or other chroniccomplement-mediated disorder comprising administering a complementinhibitor according to a dosing schedule that includes an inductionphase followed by a maintenance phase, wherein the maintenance phasecomprises treating the subject according to the method of claim 1, andwherein the complement inhibitor is administered (i) at a higher doseduring the induction phase than during the maintenance phase; (ii) morefrequently during the induction phase than during the maintenance phase;or (iii) at a higher dose and more frequently during the induction phasethan during the maintenance phase.
 22. The method of claim 1, whereinthe successive doses are administered at intervals equal to at least 5times the terminal plasma half-life of the complement inhibitor whenadministered by the same route.
 23. The method of claim 1, wherein saidhalf-life is between 1 and 5 days, between 5 and 10 days, between 10 and20 days, or between 20 and 30 days.